Method of treating an inflammatory or infectious disease

ABSTRACT

A method of treating an inflammatory or infectious disease includes administering an effective amount of pharmaceutical composition to a subject in need of treatment of the inflammatory or infectious disease. The composition includes an aqueous suspension of nanoparticles of a glucocorticosteroid compound.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to an aqueous suspension containingglucocorticosteroid nanoparticles and the use thereof.

Description of the Related Art

Glucocorticosteroids are hydrophobic and have been provided in the formof aqueous suspensions. However, the aqueous suspension of aglucocorticosteroid compound have a problem that the contained steroidparticles precipitate as time advances, and thus a patient needs toshake a container before use to disperse an active componenthomogeneously in the liquid phase. Even in case that a patient shook acontainer before use without fail, the particles in the suspensioneasily agglomerate to form cluster, thereby the particle diameter of thedrug increases and the uniformity is lost. Such a ununiform dispersioncaused a loss in an administration dose that is predetermined andconsequent insufficient suppression of inflammation and pains.

In order to solve such a problem caused by the steroid, emulsionpreparations have been proposed (Patent Literature 1, Non PatentLiteratures 1, 2) as one of the methods. For example, Difluprednate O/Wemulsion preparation (Durezol (Registered trademark): a 0.05%difluprednate preparation) has been confirmed to be stably applied toaffected area with a uniform drug regardless of storage conditions orshaking before use.

However, the O/W emulsion preparation requires to use an oil solvent,which causes a problem of irritating effects such as uncomfortablesensations or congestion. Thus, it has been required to prepareglucocorticosteroid aqueous preparations without using oil solvent thatcan maintain the uniformity.

Modification of structure which gives hydrophilicity to the compound,such as Dexamethasone sodium phosphate, has been attempted to dissolvethe compound in water. However, the water dissolved preparations couldcontain limited concentration of an active component due to the poorsolubility.

As alternative aqueous solution containing a hardly soluble drug,nanosuspensions which contains nano-sized particles of an activecomponent in an aqueous suspension have been proposed. It has been knownthat the particle diameter as small as nanometer substantially extendsspecific surface area in the nanosuspensions, and this enables fastermaximization of the serum level of the component due to its increasedsolubility, variety of administration forms, and higher amount of anactive component to be contained. As the nanosuspension of aglucocorticosteroid compound, it has been disclosed that the aqueoussuspension containing fluticasone (D90 0.4 μm) and budesonide (D90 0.4μm) produced by a wet mill using glass beads maintained the uniformity,crystal structure, and particle diameter after five weeks preservationat 4° C. (Non Patent Literature 3). Another approach for formingnanoparticles as bottom-up approach has been reported that precipitateshydrocortisone, a glucocorticosteroid compound, so as to generatenanoparticles having a mean diameter of about 300 nm, which is preparedas an aqueous suspension (Non Patent Literature 4). However, they alsoreported that the top-down approach (milling) is more advantageous inboth intraocular pressure elevation and stability. Anothernanosuspension containing a corticosteroid (specifically, mometasonefuroate) mainly used for transnasal administration has been disclosedwhich contains corticosteroid having a D50 of 50 to 500 nm, ahydrophilic polymer, a wetting agent, and a complexing agent (PatentLiterature 2). Additionally, an autoclave-sterilizable aqueoussuspension of a glucocorticosteroid compound has been reported (PatentLiterature 3).

CITATION LIST Patent Literature

-   [Patent Literature 1] International Publication No. WO 97/05882-   [Patent Literature 2] U.S. Unexamined Patent Application Publication    No. 2011/0008453-   [Patent Literature 3] International Publication No. WO 2007/089490

Non Patent Literature

-   [Non Patent Literature 1] Eric D Donnenfeld, Clinical    Opthalmology (2011) 5:811-816-   [Non Patent Literature 2] Hetal K. Patel et al., Colloids and    Surfaces: Biointerfaces (2013) 102:86-94-   [Non Patent Literature 3] Jerry Z. Yang et al., Journal of    Pharmaceutical Sciences (2008) 97 (11):4869-4878-   [Non Patent Literature 4] Hany S. M. Ali et al., Journal of    Controlled Release (2011) 149:175-181

SUMMARY OF THE INVENTION Technical Problem

Despite extensive studies on aqueous solutions containing such hardlysoluble agents, it is still difficult to practically use aqueoussuspensions such as injections, eye drops and ear drops containing aglucocorticosteroid compound such as clobetasol propionate. It has thusbeen expected to develop aqueous suspensions for injections and fortopical administrations, specifically eye drops and ear drops,containing a glucocorticosteroid compound as the active component withgood temporal stability and dispersion stability.

Accordingly, in one embodiment, the present invention is aimed toprovide an aqueous suspension containing as an active component aglucocorticosteroid compound, which has good temporal stability anddispersion stability. More specifically, the present invention is aimedto provide aqueous pharmaceutical compositions such as injections, eyedrops, ear drops, nose drops, and/or inhalers containing aglucocorticosteroid compound as the active component, which has goodtransparency, dispersibility, and storage stability. The further objectof the invention is to provide an eye drop containing aglucocorticosteroid compound as an active component, which is highretention in the cornea and good transferability into the aqueous humor.The present invention is also objected to provide an aqueous suspensionor an aqueous pharmaceutical composition containing clobetasolpropionate, a glucocorticosteroid compound, as the active component.

Solution to Problems

The inventors conducted extensive studies and found that the aqueoussuspension containing nanoparticles of a glucocorticosteroid compoundand, if necessary, a dispersion stabilizer, a surfactant, anagglomeration inhibitor and/or a viscosity modifier is excellent intransparency, (long term) dispersibility, storage stability, retentionin the cornea, and transferability into the aqueous humor, and thus isuseful for aqueous pharmaceutical composition. The inventors found thatthe aqueous suspension containing nanoparticles of a glucocorticosteroidcompound and, if necessary, a dispersion stabilizer, a surfactant, anagglomeration inhibitor and/or a viscosity modifier exceptionallyachieves good transparency, (long term) dispersibility and storagestability without containing an organic compound that causes irritatingeffects such as uncomfortable sensations or congestion. With thesefindings, the inventors have accomplished the highly effectiveanti-inflammatory aqueous preparation containing a glucocorticosteroidcompound which can stably provide a uniform drug to an affected sitewith less irritation.

The present invention more specifically relates to the followings:

(1) An aqueous suspension containing nanoparticles of aglucocorticosteroid compound.

(2) The aqueous suspension of (1), wherein a mean particle diameter ofthe nanoparticles is 300 nm or less and a D90 particle diameter of thenanoparticles is 450 nm or less.

(3) The aqueous suspension of (1) or (2), wherein the nanoparticles areproduced by mixing a glucocorticosteroid compound, a physiologicallyacceptable salt, a physiologically acceptable polyol, and a surfacemodifier.

(4) The aqueous suspension of any one of (1) to (3), wherein theglucocorticosteroid compound is one or more substances selected fromclobetasol propionate, diflorasone diacetate, dexamethasone propionate,difluprednate, mometasone furoate, diflucortolone valerate,betamethasone butyrate propionate, fluocinonide, hydrocortisone butyratepropionate, beclomethasone dipropionate, deprodone propionate,betamethasone valerate, dexamethasone valerate, prednisolone valerateacetate, fluocinolone acetonide, hydrocortisone butyrate, clobetasonebutyrate, alclometasone dipropionate, triamcinolone acetonide,flumethasone pivalate, prednisolone, and hydrocortisone.(5) The aqueous suspension of any one of (1) to (4), further containinga dispersion stabilizer.(6) The aqueous suspension of (5), wherein the dispersion stabilizer ispolyoxyethylene polyoxypropylene glycol and/or polyvinyl alcohol.(7) The aqueous suspension of any one of (1) to (6), further containinga viscosity modifier.(8) The aqueous suspension of (7), wherein the viscosity modifier is oneor more substances selected from methylcellulose,hydroxypropylmethylcellulose and polyvinylalcohol.(9) The aqueous suspension of (7) or (8), containing 1 to 10 mg/mL ofthe viscosity modifier.(10) A pharmaceutical composition containing the aqueous suspension ofany one of (1) to (9).(11) The pharmaceutical composition of (10), which is for parenteraladministration.(12) The pharmaceutical composition of (11), which is for an injectionor for a topical preparation.(13) The pharmaceutical composition of (12), which is for a topicalpreparation for an eye, an ear, a nose or a lung.(14) The pharmaceutical composition of (13), which is an eye drop, anear drop, a nose drop, or an inhaler.(15) The pharmaceutical composition of any one of (10) to (14), which isa therapeutic agent or a preventive agent for an inflammatory orinfectious disease.(16) The pharmaceutical composition of (15), wherein the inflammatory orinfectious disease is a systemic inflammatory or infectious disease.(17) The pharmaceutical composition of (15), wherein the inflammatory orinfectious disease is a topical inflammatory or infectious disease.(18) The pharmaceutical composition of (17), wherein the topical area isone or more tissues or organs selected from eyes, ears, nose (upperrespiratory tract), and lungs (lower respiratory tract).(19) A kit for preparing the pharmaceutical composition of any one of(10) to (18), comprising nanoparticles of a glucocorticosteroidcompound.(20) A method for manufacturing the pharmaceutical composition of anyone of (10) to (18), comprising mixing a glucocorticosteroid compound, aphysiologically acceptable salt, a physiologically acceptable polyoland/or water, and a dispersion stabilizer.(21) The method for manufacturing of (20), comprising mixing aglucocorticosteroid compound, a physiologically acceptable salt,glycerin, anhydrous citric acid, and hydrogenated soybean lecithin.

Particularly, the inventors found that nanoparticles of aglucocorticosteroid compound have excellent transferability into theaqueous humor and an good anti-inflammatory action, when thenanoparticles have a mean particle diameter (hereinafter referred to as“Dv”) of 300 nm or less and a 90% diameter (hereinafter referred to as“D90”) of 450 nm or less (preferably, a Dv of 250 nm or less and a D90of 300 nm or less, or a Dv of 200 nm or less and a D90 of 250 nm orless). Employing such nanoparticles, the solubility of theglucocorticosteroid compound is expected to become higher, whichincreases the bioavailability and reduces the administration dose. Themean particle diameter can be measured as intensity distribution meanparticle diameter, volume distribution mean particle diameter, andnumber distribution mean particle diameter. The Dv herein preferablyrepresents the intensity distribution mean particle diameter.

The present invention thus relates to, in one embodiment, the aqueoussuspension containing nanoparticles of a glucocorticosteroid compound,and preferably to the aqueous suspension wherein the nanoparticles has aDv of 300 nm or less and a D90 of 450 nm or less. The aqueous suspensioncontains, for example, nanoparticles of a glucocorticosteroid compoundproduced by mixing a glucocorticosteroid compound, a physiologicallyacceptable salt, a physiologically acceptable polyol and/or water and adispersion stabilizer. The aqueous suspension more preferably containsnanoparticles of a glucocorticosteroid compound produced by mixing aglucocorticosteroid compound, a physiologically acceptable salt,glycerin, anhydrous citric acid and hydrogenated soybean lecithin.

The inventors additionally found that the aqueous suspension containingnanoparticles of a glucocorticosteroid compound exhibits good long-termtransparency, dispersibility, and storage stability, containing withpolyoxyethylene polyoxypropylene glycols (hereinafter referred to as“POE-POP glycol”) and/or polyvinyl alcohols (hereinafter referred to as“PVA”) as a dispersion stabilizer, and/or containing with hydroxypropylmethylcellulose and/or methyl cellulose as a thickener.

The present invention thus relates to, in one embodiment, the aqueoussuspension containing nanoparticles of a glucocorticosteroid compoundhaving a Dv of 300 nm or less and a D90 of 450 nm or less (preferably, aDv is 250 nm or less and a D90 is 300 nm or less, or a Dv is 200 nm orless and a D90 is 250 nm or less). The present invention relates to, inanother embodiment, the aqueous pharmaceutical composition containingnanoparticles of a glucocorticosteroid compound as an effective agentand a dispersion stabilizer and/or a viscosity modifier as an additive.

The “aqueous pharmaceutical composition” herein means an aqueous liquidor gel pharmaceutical composition, specifically a pharmaceuticalcomposition containing nanoparticles of a glucocorticosteroid compoundsuspended in the aqueous liquid or gel. The pharmaceutical compositionherein accordingly means an aqueous pharmaceutical composition unlessotherwise stated. The aqueous pharmaceutical composition includesinjections and topical preparations. The topical preparations hereinaccordingly mean aqueous preparations for topical administrations. Theaqueous pharmaceutical composition may be viscous as long as notpreventing the composition from using as a pharmaceutical drug, andincludes gel preparations as well as watery preparations.

The “topical area” herein means a part of the body, including anaffected site, an area around the affected site or an organ includingthe affected site, and preferably is the eye, ear, nose (upperrespiratory tract) or lung (lower respiratory tract).

The injection may be for treating or preventing a systemic or topicalinflammatory or infectious disease, and includes injections such asintravenous injections, subcutaneous injections, intramuscularinjections, and intravenous drips.

The “topical preparation” herein means a pharmaceutical compositionaimed to be administered locally. The topical preparation preferablyincludes topical eye preparations (e.g., eye drops), topical earpreparations (e.g., ear drops), topical nose preparations (e.g., nosedrops) and topical lung preparations (e.g., inhalers). These topicalpreparations can be used to treat or prevent inflammatory or infectiousdiseases of the eye, ear, nose or lung. The preparation form alsoincludes eye drops, ear drops, nose drops and inhalers. The topicalpreparations may preferably be topical eye preparations (including eyedrops) for treating or preventing ocular inflammatory or infectiousdiseases, topical ear preparations (including ear drops) for treating orpreventing otogenic inflammatory or infectious diseases, topical nosepreparations (including nose drops) for treating or preventing nasalinflammatory or infectious diseases or topical lung preparations(including inhalers) for treating or preventing pulmonary inflammatoryor infectious diseases.

The aqueous pharmaceutical composition can be used to treat or preventinflammatory or infectious diseases by topically administering aneffective amount thereof to a patient in need thereof. In other words,the present invention relates to, in one embodiment, a method fortreatment or prevention of inflammatory or infectious diseasescomprising administering an effective amount of the aqueous suspensionor the pharmaceutical composition containing the aqueous suspension,wherein the aqueous suspension or pharmaceutical composition containsnanoparticles of a glucocorticosteroid compound and optionally adispersion stabilizer and/or a viscosity modifier to a patient in needthereof. The present invention, for example, encompasses a method fortreatment or prevention of inflammatory or infectious diseasescomprising topically administering an effective amount of the topicalpreparation containing nanoparticles of a glucocorticosteroid compoundand optionally a dispersion stabilizer to a patient in need thereof.

Alternatively, the present invention relates to a use of nanoparticlesof a glucocorticosteroid compound (optionally with a dispersionstabilizer and/or a viscosity modifier) or a use of an aqueoussuspension containing said nanoparticles, for manufacturing an aqueouspharmaceutical composition (e.g., injections and topical preparations).

The “glucocorticosteroid compound” herein is not limited as long as itis glucocorticosteroid and derivatives thereof. Examples of theglucocorticosteroid compound include clobetasol propionate, diflorasonediacetate, dexamethasone propionate, difluprednate, mometasone furoate,diflucortolone valerate, betamethasone butyrate propionate,fluocinonide, hydrocortisone butyrate propionate, beclomethasonedipropionate, deprodone propionate, betamethasone valerate,dexamethasone valerate, prednisolone valerate acetate, fluocinoloneacetonide, hydrocortisone butyrate, clobetasone butyrate, alclometasonedipropionate, triamcinolone acetonide, flumethasone pivalate,prednisolone and hydrocortisone, and clobetasol propionate ispreferable.

The “aqueous suspension” herein means an aqueous liquid in whichnanoparticles of a glucocorticosteroid compound are suspended. Theaqueous suspension herein may constitute a pharmaceutical compositionwhich can be administered as a pharmaceutical drug by itself, or mayconstitute a pharmaceutical composition by adding other components and adiluent (e.g. raw materials for pharmaceutical composition), or may notbe used for a pharmaceutical drug.

The aqueous suspension herein includes dispersion-stabilized aqueoussuspensions. The dispersion-stabilized means that the aqueous suspensionhas any one of, or two or more of, the properties of (1) noprecipitation confirmed under visual inspection, (2) high transparency,(3) no agglomerate or crystal observed under microscopic observation,and (4) no substantial changes in the Dv value (not 50% or moreincrease) after dispersion by stirring followed by standing for 24 hours(preferably 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 1 year or 2 years) atroom temperature (25° C.) The aqueous suspension containingnanoparticles of a glucocorticosteroid compound herein is preferably anaqueous suspension with no precipitation confirmed under visualinspection, high transparency, and no agglomerate or crystal observedunder microscopic observation after 7 days from sealed in a test tube.

The transparency can be determined in conformity with the transparencytest described in The Japanese Pharmacopoeia. Specifically, thetransparency can be determined by the following procedures. Water isadded to 5 mL of a formazine standard up to 100 mL, which is used as aturbidity standard. Each of a tested aqueous suspension and a newlyprepared turbidity standard is taken to a colorless clear glassflat-bottom test tube having an inner diameter of 15 mm such that theliquid layer has a depth of 30 mm or 40 mm, which is then compared eachother by observing from above on a black backdrop in the scatteringlight. When the transparency of the tested aqueous suspension is thesame as water or the solvent used, or when the turbidity of the testedaqueous suspension is lower than the turbidity standard, thetransparency is determined to be high. Alternatively, transmittances at660 nm of a tested aqueous suspension and of a newly prepared turbiditystandard are measured by the ultraviolet visible spectrophotometrymethod using a 50 mm layer cell, with using water or the solvent as thecontrol. When the transmittance of the tested aqueous suspension ishigher than the turbidity standard, the transparency of the testedaqueous suspension is determined to be high.

In another embodiment, the topical preparation is a topical eyepreparation having transferability into the aqueous humor. The“transferability into the aqueous humor” herein means that aconcentration of a glucocorticosteroid compound (average value) in theaqueous humor is 45 ng/mL or more (preferably 50 ng/mL or more, 55 ng/mLor more, 60 ng/mL or more, 65 ng/mL or more, 70 ng/mL or more, 75 ng/mLor more) after 60 minutes from a single eye drop administration of theaqueous topical preparation containing a glucocorticosteroid compoundadjusted to be 0.05% (w/v). The “transferability into the aqueous humor”alternatively may mean that a concentration of glucocorticosteroidcompound (average value) in the aqueous humor is 40 ng/mL or more(preferably 50 ng/mL or more, 55 ng/mL or more, 60 ng/mL or more, 63ng/mL or more, 64 ng/mL or more, 65 ng/mL or more, 70 ng/mL or more, 75ng/mL or more) after 30 minutes from a single eye drop administration ofthe aqueous topical preparation containing a glucocorticosteroidcompound adjusted to be 0.05% (w/v).

In another embodiment, the topical preparation is a topical eyepreparation having transferability into the conjunctiva. The“transferability into the conjunctiva” herein means that a concentrationof a glucocorticosteroid compound (average value) in the conjunctiva is500 ng/mL or more (preferably 659 ng/mL or more, 900 ng/mL or more, 972ng/mL or more, 1000 ng/mL or more, 1200 ng/mL or more, 1210 ng/mL ormore, 1400 ng/mL or more, 1455 ng/mL or more, 1500 ng/mL or more or 2000ng/mL or more, 2141 ng/mL or more) after 15 minutes from a single eyedrop administration of the aqueous topical preparation containing aglucocorticosteroid compound adjusted to be 0.05% (w/v).

The transferability into the aqueous humor and the conjunctiva can bedetermined according to the method described in the Examples of thisapplication by using appropriate animals, and for example by thefollowing procedures. The lower eyelid of a rabbit is gently pulled off,an eye drop of the test substance is administered (a single eye dropadministration) into the conjunctival sac of the left eye using apipette, and after administration, the upper and lower eyelids aregently closed and held for about 2 seconds. After 15 minutes, 30minutes, 60 minutes and 90 minutes from the administration, the rabbitsare anesthetized and euthanized by bleeding, followed by thoroughlywashing the eye with water for injection, and the aqueous humor andconjunctiva are collected. A concentration of glucocorticosteroidcompound in the collected aqueous humor can be determined by addingmethanol and an internal standard (prednisolone) solution to thecollected aqueous humor, stirring the mixture, subsequently addingacetonitrile thereto, stirring the mixture, and centrifuging (13,100×g,4° C., 5 minutes) the mixture, followed by measuring the supernatantobtained by centrifuge by the LC-MS/MS method. A concentration of theglucocorticosteroid compound in the collected conjunctiva can bedetermined by adding ultrapure water in nine fold volume of the wetweight of the obtained conjunctiva, homogenizing, further addingmethanol and an internal standard (prednisolone) solution thereto,stirring the mixture, subsequently adding acetonitrile thereto, stirringthe mixture, and centrifuging the mixture (13100×g, 4° C., 5 minutes),followed by measuring the supernatant obtained by centrifuge by theLC-MS/MS method.

In another embodiment, the topical preparation is a topical eyepreparation capable of reducing an increase rate of proteinconcentration in the aqueous humor. Being “capable of reducing anincrease rate of protein concentration in the aqueous humor” means thata protein concentration in the aqueous humor which is obtained byadministering 40 μL of the aqueous topical preparation containing a0.05% (w/v) or 0.1% (w/v) glucocorticosteroid compound seven times at30-60 minutes intervals before and after keratocentesis (preferably,setting the time of keratocentesis as 0 minutes, seven administrationsat 180 minutes, 120 minutes, 60 minutes and 30 minutes before thekeratocentesis, and 30 minutes, 60 minutes and 90 minutes after thekeratocentesis) to an experimental animal (e.g., rabbit) and collectingthe aqueous humor after 30 minutes from the final administration, isless than three times (preferably less than 2.5 times or less than twotimes) of the protein concentration in the aqueous humor of the eye towhich keratocentesis is not carried out.

In another embodiment, the topical preparation is a topical eyepreparation capable of inhibiting an inflammation of the eye.Specifically, the topical preparation is a topical eye preparationcapable of suppressing a production of prostaglandin E2 (PGE2) that isan inflammation mediator. Being “capable of suppressing a production ofPGE2” means that a PGE2 concentration in the aqueous humor which isobtained by administering 40 μL of the aqueous topical preparationcontaining a 0.05% (w/v) or 0.1% (w/v) glucocorticosteroid compoundseven times at 30-60 minute intervals before and after keratocentesis(preferably, setting the time of keratocentesis as 0 minutes, sevenadministrations at 180 minutes, 120 minutes, 60 minutes and 30 minutesbefore the keratocentesis, and 30 minutes, 60 minutes and 90 minutesafter the keratocentesis) to an experimental animal (e.g., rabbit) andcollecting the aqueous humor after 30 minutes from the finaladministration, is lower than the PGE2 concentration in the aqueoushumor obtained by the same manner with administering Durezol (Registeredtrademark).

The topical eye preparation may have two or more (two, three or all)properties selected from the transferability into the aqueous humor, thetransferability into the conjunctiva, the reduction of increase rate ofa protein concentration in the aqueous humor and the inflammationinhibitory activity on the eye.

In one embodiment, the aqueous suspension is an aqueous suspension withlow irritability. The low irritability herein means that a degree ofirritating reactions (inflammation reactions such as flare, swellingand/or congestion) in administering the aqueous suspension to a subjectis lower than that in administering previously used aqueous preparationscontaining the same active component. Whether the irritability of a testaqueous suspension is low or not can be determined, for example, withreferring to the method of Jonas, J. Kuehne et al., Am J Ophthalmol(2004) 138:547-553, by administering the test aqueous suspension to theeye of a rabbit, measuring the degree of eye inflammation, anddetermining that the irritability is low, when the degree ofinflammation is lower than the standard liquid agent (the same asabove). More specifically, in case of an eye drop, the irritability isdetermined by applying a preparation containing 1.0% glucocorticosteroidcompound to the eye once to 20 times a day at intervals of 30 minutes toseveral hours, observing the cornea, iris and conjunctiva beforeadministration and 1, 3, 5 and 24 hours after the final administration,and scoring in accordance with Draize's scoring criteria (see OECDGUIDELINES FOR TESTING OF CHEMICALS 405 (24 Feb. 1987) Acute EyeIrritation/Corrosion).

The aqueous suspension or pharmaceutical composition may contain one ortwo or more physiologically acceptable salts. Examples of the“physiologically acceptable salt” include sodium chloride, potassiumchloride, ammonium chloride, sodium sulfate, magnesium sulfate,potassium sulfate, calcium sulfate, sodium malate, sodium citrate,disodium citrate, sodium dihydrogen citrate, potassium dihydrogencitrate, sodium dihydrogen phosphate, potassium dihydrogen phosphate,disodium hydrogen phosphate, and dipotassium hydrogen phosphate, andsodium chloride is preferable.

The aqueous suspension or pharmaceutical composition can contain thephysiologically acceptable salt at a concentration of 0.01 to 10%,preferably 0.1 to 5% or, for example, 0.5 to 3%, 0.8 to 2%.Alternatively, the aqueous suspension or pharmaceutical composition cancontain the physiologically acceptable salt at a concentration of 0.01to 50 mg/mL, 0.1 to 20 mg/mL or 1 to 5 mg/mL.

The aqueous suspension or pharmaceutical composition may contain one ortwo or more surfactants and/or one or two or more agglomerationinhibitors.

The “surfactant” is not limited as long as it can be administered to ahuman as a pharmaceutical additive without showing toxicity and withouthindering the activity of glucocorticosteroid compound. The surfactantmay be, for example, non-ionic surfactant including polyoxyethylene(hereinafter referred to as “POE”)-polyoxypropylene (hereinafterreferred to as “POP”) block copolymers such as poloxamer 407, poloxamer235 and poloxamer 188; ethylenediamine adducts topolyoxyethylene-polyoxypropylene block copolymer such as poloxamine; POEsorbitan fatty acid esters such as POE (20) sorbitan monolaurate(polysorbate 20), POE (20) sorbitan monooleate (polysorbate 80) andpolysorbate 60; POE hydrogenated castor oils such as POE (60)hydrogenated castor oil; POE alkyl ethers such as POE (9) lauryl ether;POE-POP alkyl ethers such as POE (20) POP (4) cetyl ether; POEalkylphenyl ethers such as POE (10) nonyl phenyl ether; POE-POP glycolssuch as POE (105) POP (5) glycol, POE (120) POP (40) glycol, POE (160)POP (30) glycol, POE (20) POP (20) glycol, POE (200) POP (70) glycol,POE (3) POP (17) glycol, POE (42) POP (67) glycol, POE (54) POP (39)glycol and POE (196) POP (67) glycol; amphoteric surfactants includingglycine-type surfactants such as alkyldiaminoethyl glycine, betaineacetate-type surfactants such as lauryl dimethylaminoacetic acidbetaine, and imidazoline-type surfactants; anionic surfactants includingPOE alkyl ether phosphates and salts thereof such as POE (10) sodiumlauryl ether phosphate, N-acylamino acid salts such as sodium lauroylmethyl alanine, alkyl ether carboxylates, N-acyl taurates such as sodiumcocoyl N-methyltaurate, sulfonates such as sodium tetradecenesulfonate,alkyl sulfates such as sodium lauryl sulfate, POE alkyl ether sulfatessuch as POE (3) sodium lauryl ether sulfate, and α-olefin sulfonates;and cationic surfactants including alkylamine salts, alkyl quarternaryammonium salts (benzalkonium chloride and benzethonium chloride) andalkyl pyridinium salts (cetylpyridinium chloride and cetylpyridiniumbromide). The aqueous suspension may contain one or two or moresurfactants.

The “agglomeration inhibitor” herein is not limited as long as itinhibits an agglomeration of the glucocorticosteroid compound and it canbe administered to a human without showing toxicity and withouthindering the activity of glucocorticosteroid compound. Theagglomeration inhibitor may be phospholipids such as alkyl sulfate,N-alkyloyl methyl taurate, ethanol, glycerol, propylene glycol, sodiumcitrate, phospholipids including glycerophospholipid (lecithin(phosphatidylcholine) (e.g., refined soybean lecithin, hydrogenatedsoybean lecithin), phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, phosphatidic acid, phosphatidylglycerol,lysophosphatidylcholine, lysophosphatidylserine,lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidicacid and lysophosphatidylglycerol) and sphingophospholipids(sphingomyelin, ceramide, glycosphingolipid or ganglioside), D-sorbitol,lactose, xylitol, gum arabic, sucrose fatty acid ester, polyoxyethylenehydrogenated castor oil, polyoxyethylene fatty acid esters,polyethyleneglycol (PEG), polyoxyethylene sorbitan fatty acid ester,alkyl benzene sulfonate, sulfosuccinate, POE-POP glycol,polyvinylpyrrolidone, PVA, hydroxypropyl cellulose, methyl cellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, carmellosesodium, carboxyvinyl polymers, N-acyl-glutamate, acrylic acidcopolymers, methacrylic acid copolymers, casein sodium, L-valine,L-leucine, L-isoleucine, benzalkonium chloride and benzethoniumchloride. The aqueous suspension may contain one agglomeration inhibitoror two or more agglomeration inhibitors.

The aqueous suspension or pharmaceutical composition can containagglomeration inhibitor at a concentration of 0.001 to 10% or 0.01 to10%, preferably 0.02 to 5%, for example, 0.03 to 1%, 0.04 to 0.5%, 0.05to 0.2%. Alternatively, the aqueous suspension or pharmaceuticalcomposition can contain the agglomeration inhibitor at a concentrationof 0.01 to 50 mg/mL, 0.1 to 20 mg/mL or 1 to 5 mg/mL.

The surfactant and/or the agglomeration inhibitor are preferably one ormore substances selected from polyoxyethylene hydrogenated castor oil 60(e.g., HCO-60), polyoxyethylene hydrogenated castor oil 40 (e.g.,HCO-40), polysorbate 80 (e.g., Tween 80), polysorbate 20 (e.g., Tween20), POE-POP glycol (e.g., PLONON 407P, Pluronic F68, UNILUB 70DP-950B)and PVA (e.g., Kuraray POVAL 217C), and more preferably one or moresubstances selected from POE-POP glycol and PVA.

The “viscosity modifier” herein is not limited as long as it is capableof adjusting the viscosity of the aqueous suspension and it can beadministered to a human as a pharmaceutical additive without showingtoxicity and without hindering the activity of the glucocorticosteroidcompound. The viscosity modifier may be polysaccharides or derivativesthereof (gum arabic, gum karaya, xanthan gum, carob gum, guar gum, gumguaiac, quince seed, darman gum, gum tragacanth, benzoin rubber, locustbean gum, casein, agar, alginic acid, dextrin, dextran, carrageenan,gelatin, collagen, pectin, starch, polygalacturonic acid, chitin andderivatives thereof, chitosan and derivatives thereof, elastin, heparin,heparinoid, heparin sulfate, heparan sulfate, hyaluronic acid andchondroitin sulfate), ceramide, cellulose derivatives (methyl cellulose,ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, carboxymethyl cellulose, carboxyethylcellulose, cellulose and nitrocellulose), PVA (completely or partiallysaponified), polyvinylpyrrolidone, Macrogol, polyvinyl methacrylate,polyacrylic acid, carboxyvinyl polymer, polyethyleneimine, polyethyleneoxide, polyethylene glycol, ribonucleic acid, deoxyribonucleic acid,methyl vinyl ether-maleic anhydride copolymers, and pharmacologicallyacceptable salts thereof (e.g., sodium alginate). The aqueous suspensionmay contain one or two or more viscosity modifiers. The viscositymodifier is preferably one or more substances selected fromhydroxypropyl methylcellulose (e.g., TC-5®, Metlose 60SH-50), PVA(Kurary POVAL 217C) and methyl cellulose (e.g., Metlose SM-100, MetloseSM-15), with one or more substances selected from hydroxypropylmethylcellulose and methyl cellulose being more preferable.

The aqueous suspension can contain 1 to 10 mg/mL, preferably 1 to 5mg/mL, for example, 1 to 4 mg/mL, 1 to 3 mg/mL, 1 to 2 mg/mL, of theviscosity modifier.

The dispersion stabilizer usable herein is the substances listed aboveas the surfactants, agglomeration inhibitors and/or viscosity modifiers,and is preferably one or more substances selected from polyoxyethylenehydrogenated castor oil 60, polyoxyethylene hydrogenated castor oil 40,polysorbate 80, polysorbate 20, POE-POP glycol, PVA, hydroxypropylmethylcellulose and methyl cellulose, and more preferably one or moresubstances selected from POE-POP glycol, PVA, hydroxypropylmethylcellulose and methyl cellulose.

The surfactant, agglomeration inhibitor and/or viscosity modifier whichare also used as the dispersion stabilizer (hereinafter referred to as“additives” in this paragraph) may adhere to or be adsorbed on thesurface of nanoparticles of a glucocorticosteroid compound. When theseadditives are added before the pulverization step, these additivesadhere to or are adsorbed on the surface of nanoparticles of aglucocorticosteroid compound, which results in inhibiting thenanoparticle agglomeration during the pulverization step. By adhering toor by adsorbing on the surface of nanoparticles of a glucocorticosteroidcompound, the additives effectively inhibit the agglomeration in theaqueous suspension. In this context, the surfactant, agglomerationinhibitor and/or viscosity modifier which can be also used as thedispersion stabilizer can be construed to adhere to or to be adsorbed onthe surface of nanoparticles of a glucocorticosteroid compound, as longas at least a part of the additives adheres to or is adsorbed on thenanoparticle surface (contributing to the surface modification), and itis not necessary that the additive neither adhering nor being adsorbedis not present in the aqueous suspension. The “surface modifier” hereinrefers to the surfactant, the agglomeration inhibitor and/or theviscosity modifier which can be the dispersion stabilizer, which iscapable of modifying the nanoparticle surface of a glucocorticosteroidcompound.

The aqueous suspension or pharmaceutical composition may contain one ortwo or more physiologically acceptable polyols. The pharmaceuticalcomposition may contain, for example, the physiologically acceptablepolyols described above. The “physiologically acceptable polyols”include glycerin, propylene glycol, polyethylene glycol, dipropyleneglycol and diethylene glycol, and preferably is propylene glycol orglycerin. The aqueous suspension or pharmaceutical composition cancontain the physiologically acceptable polyol at a concentration of, forexample, 0.001 to 10% or 0.01 to 10%, preferably, 0.02 to 5%, forexample, 0.03 to 1%, 0.04 to 0.5%, 0.05 to 0.2%. Alternatively, theaqueous suspension or pharmaceutical composition can contain thephysiologically acceptable polyol at a concentration of 0.01 to 10mg/mL, 0.05 to 5 mg/mL or 0.1 to 3 mg/mL.

The aqueous suspension or aqueous pharmaceutical composition does notcontain an oil solvent. The oil solvent means a water-insoluble orslightly water-soluble solvent.

The glucocorticosteroid compound contained in the aqueous suspension oraqueous pharmaceutical composition is in the form of nanoparticles. Themean particle diameter (Dv) of the glucocorticosteroid compoundnanoparticles may be 300 nm or less, preferably 250 nm or less, 240 nmor less, 230 nm or less, 220 nm or less, 210 nm or less, 200 nm or less,190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nmor less, 140 nm or less, 130 nm or less, 120 nm or less or 110 nm orless. The ranges of mean particle diameter of the glucocorticosteroidcompound may be, for example, 50 to 300 nm, 50 to 250 nm, 50 to 240 nm,50 to 230 nm, 50 to 220 nm, 50 to 210 nm, 50 to 200 nm, 50 to 190 nm, 50to 180 nm, 50 to 170 nm, 50 to 160 nm, 50 to 150 nm, 50 to 140 nm, 50 to130 nm, 50 to 120 nm, 50 to 110 nm, 100 to 300 nm, 100 to 250 nm, 100 to240 nm, 100 to 230 nm, 100 to 220 nm, 100 to 210 nm, 100 to 200 nm, 100to 190 nm, 100 to 180 nm, 100 to 170 nm, 100 to 160 nm, 100 to 150 nm,100 to 140 nm, 100 to 130 nm, 100 to 120 nm or 100 to 110 nm.

The 90% diameter (D90) of the glucocorticosteroid compound nanoparticlescontained in the aqueous suspension or aqueous pharmaceuticalcomposition is 450 nm or less, preferably 400 nm or less, 350 nm orless, 300 nm or less, 290 nm or less, 280 nm or less, 270 nm or less,260 nm or less, 250 nm or less, 240 nm or less or 230 nm or less. Theranges of 90% diameter (D90) of the glucocorticosteroid compound may be,for example, 50 to 400 nm, 50 to 350 nm, 50 to 300 nm, 50 to 290 nm, 50to 280 nm, 50 to 270 nm, 50 to 260 nm, 50 to 250 nm, 50 to 240 nm, 50 to230 nm, 100 to 400 nm, 100 to 350 nm, 100 to 300 nm, 100 to 290 nm, 100to 280 nm, 100 to 270 nm, 100 to 260 nm, 100 to 250 nm, 100 to 240 nm or100 to 230 nm.

The 50% diameter (D50) of the glucocorticosteroid compound nanoparticlescontained in the aqueous suspension or aqueous pharmaceuticalcomposition may be 200 nm or less, preferably 190 nm or less, 180 nm orless, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less,130 nm or less, 120 nm or less, 110 nm or less or 100 nm or less. Theranges of 50% diameter (D50) of the glucocorticosteroid compound may be50 to 190 nm, 50 to 180 nm, 50 to 170 nm, 50 to 160 nm, 50 to 150 nm, 50to 140 nm, 50 to 130 nm, 50 to 120 nm, 50 to 110 nm, 50 to 100 nm, 80 to190 nm, 80 to 180 nm, 80 to 170 nm, 80 to 160 nm, 80 to 150 nm, 80 to140 nm, 80 to 130 nm, 80 to 120 nm, 80 to 110 nm or 80 to 100 nm.

The glucocorticosteroid compound nanoparticles contained in the aqueoussuspension or aqueous pharmaceutical composition may meet two or moreparticle diameter conditions selected from the mean particle diameter(Dv), the 90% diameter (D90) and the 50% diameter (D50) described above.The glucocorticosteroid compound nanoparticles contained in the aqueoussuspension can have, for example, a mean particle diameter (Dv) of 166nm or less, a D50 of 138 nm or less and/or a D90 of 241 nm or less. Theglucocorticosteroid compound nanoparticles contained in the aqueouspharmaceutical composition can have, for example, a mean particlediameter (Dv) of 204 nm or less, a D50 of 177 nm or less and/or a D90 of306 nm or less.

The glucocorticosteroid compound contained in the aqueous suspension asthe active component is in the form of nanoparticles, which enables theaqueous suspension to be filter-sterilized, and thus the aqueoussuspension can be sterilized easily and hardly affecting thephysicochemical properties of the active component.

The nanoparticle of a glucocorticosteroid compound contained in theaqueous suspension is preferably those produced by mixing aglucocorticosteroid compound, a physiologically acceptable salt, aphysiologically acceptable polyol and a dispersion stabilizer. Morepreferably, the nanoparticle of a glucocorticosteroid compound is thoseproduced by mixing a glucocorticosteroid compound, a physiologicallyacceptable salt, a physiologically acceptable polyol and a dispersionstabilizer, with adding lecithin (e.g., hydrogenated soybean lecithin)during or after pulverization.

The aqueous suspension includes, for example, a preparation containing,nanoparticles of a glucocorticosteroid compound; sodium chloride;hydrogenated soybean lecithin; glycerin; anhydrous citric acid; one ormore substances selected from POE-POP glycols, polyoxyethylenehydrogenated castor oils, Polysorbate 80, PVA and POE-POP blockcopolymers; benzalkonium chloride, sorbic acid or salts thereof(potassium sorbate, sodium sorbate and triclocarban sorbate) orparaoxybenzoates (methyl parahydroxybenzoate, ethyl parahydroxybenzoate,propyl parahydroxybenzoate and butyl parahydroxybenzoate); hydroxypropylmethylcellulose and/or methyl cellulose; and sodium citrate (includingtrisodium citrate).

The aqueous suspension and the pharmaceutical composition can containwater as the main component. The pharmaceutical composition, the aqueoussuspension and/or the diluent herein may contain, as necessary, variousadditives such as a stabilizer, a flavoring agent, a thickener, asurfactant, a preservative, a disinfectant or antibacterial agent, a pHcontrol agent, a tonicity agent and a buffer.

The preservative and the disinfectant or antibacterial agent includesorbic acids or salts thereof (potassium sorbate, sodium sorbate andtriclocarban sorbate), paraoxybenzoates (methyl parahydroxybenzoate,ethyl parahydroxybenzoate, propyl parahydroxybenzoate and butylparahydroxybenzoate), acrinol, methylrosanilinium chloride, benzalkoniumchloride, benzethonium chloride, cetylpyridinium chloride,cetylpyridinium bromide, chlorhexidine or salts thereof,polyhexamethylene biguanide, alkylpolyaminoethylglycine, benzyl alcohol,phenethyl alcohol, chlorobutanol, isopropanol, ethanol, phenoxyethanol,silver supported on zirconium phosphate, mercurochrome, povidone iodine,thimerosal, dehydroacetic acid, chloroxylenol, chlorophen, resorcinol,orthophenylphenol, isopropylmethylphenol, thymol, hinokitiol, sulfamine,lysozyme, lactoferrin, triclosan, 8-hydroxyquinoline, undecylenic acid,caprylic acid, propionic acid, benzoic acid, halocarban, thiabendazole,polymyxin B, 5-chloro-2-methyl-4-isothiazolin-3-one,2-methyl-4-isothiazolin-3-one, polylysine, hydrogen peroxide,polidronium chloride, Glokill (tradename: e.g., Glokill PQ, Rhodia),polydiaryl dimethyl ammonium chloride,poly[oxyethylene(dimethyliminio)ethylene-(dimethyliminio)ethylenedichloride], polyethylene polyamine-dimethylamine epichlorohydrinpolycondensates (tradename: e.g., Busan 1157, Buckman LaboratoriesInternational, Inc.) and biguanide compounds (Cosmocil CQ (tradename,about 20 wt % content of polyhexamethylenebiguanide hydrochloride, ArchPersonal Care Products L.P.)), and pharmacologically acceptable saltsthereof. Benzalkonium chloride is preferable.

The pH control agent include inorganic acids (hydrochloric acid,sulfuric acid, phosphoric acid, polyphosphoric acid and boric acid),organic acids (lactic acid, acetic acid, citric acid, anhydrous citricacid, tartaric acid, malic acid, succinic acid, oxalic acid, gluconicacid, fumaric acid, propionic acid, aspartic acid, epsilon-aminocaproicacid, glutamic acid and aminoethylsulfonic acid), gluconolactone,ammonium acetate, inorganic bases, (sodium hydrogen carbonate, sodiumcarbonate, potassium hydroxide, sodium hydroxide, calcium hydroxide andmagnesium hydroxide), organic bases (monoethanolamine, triethanolamine,diisopropanolamine, triisopropanolamine and lysine), borax, andpharmacologically acceptable salts thereof.

The tonicity agent includes inorganic salts (sodium chloride, potassiumchloride, sodium carbonate, sodium hydrogen carbonate, calcium chloride,magnesium sulfate, sodium hydrogen phosphate, disodium hydrogenphosphate, dipotassium hydrogen phosphate, sodium thiosulfate and sodiumacetate), polyhydric alcohols (glycerin, propylene glycol, ethyleneglycol and 1,3-butylene glycol), saccharides (glucose, mannitol andsorbitol).

The buffer includes tris buffer, borate buffer, phosphate buffer,carbonate buffer, citrate buffer, acetate buffer, epsilon-aminocaproicacid and aspartate. Specific examples include boric acid or saltsthereof (sodium borate, potassium tetraborate and potassium metaborate),phosphoric acid or salts thereof (sodium hydrogen phosphate, sodiumdihydrogen phosphate and potassium dihydrogen phosphate), carbonic acidor salts thereof (sodium hydrogen carbonate and sodium carbonate),citric acid or salts thereof (sodium citrate, potassium citrate andanhydrous citric acid).

The viscosity of the aqueous suspension and the pharmaceuticalcomposition herein can be 1 to 5 mPa·s, and may be, for example, 1 to 3mPa·s.

The “%” as used herein in the composition or the content refers toweight % (w/w), unless otherwise stated.

Advantageous Effects of Invention

The aqueous suspension containing nanoparticles of theglucocorticosteroid compound of the present invention has advantages intransparency, dispersibility, storage stability, transferability intothe conjunctiva, and transferability into the aqueous humor, with lowirritability, and thus is easily sterilized and has good temporalstability and dispersion stability. The suspension can be used forpharmaceutical compositions for parenteral administration, specificallyfor eye drops.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the time course change of clobetasolpropionate concentration in aqueous humor after ophthalmicadministration of the nanosuspension eye drop prepared in Examples 5(1)to 5(3). The ordinate represents the clobetasol propionate concentration(ng/mL) in aqueous humor, and the abscissa represents the elapsed time(minutes) after the administration. The black circles indicate a 0.05%ophthalmic nanosuspension (mean particle diameter 100 nm), the blacksquares indicate a 0.05% ophthalmic nanosuspension (mean particlediameter 300 nm), and the black triangles indicate a 0.05% ophthalmicnanosuspension (mean particle diameter 600 nm). The indicated values areaverage, and the error bars indicate standard deviations.

FIG. 2 is a graph showing the time course change of clobetasolpropionate concentration in conjunctiva after ophthalmic administrationof the nanosuspension eye drop prepared in Examples 5(1) to 5(3). Theordinate represents the clobetasol propionate concentration (ng/mL) inconjunctiva, and the abscissa represents the elapsed time (minutes)after the administration. The black circles indicate a 0.05% ophthalmicnanosuspension (mean particle diameter 100 nm), the black squaresindicate a 0.05% ophthalmic nanosuspension (mean particle diameter 300nm), and the black triangles indicate a 0.05% ophthalmic nanosuspension(mean particle diameter 600 nm). The indicated values are average, andthe error bars indicate standard deviations.

FIG. 3 is a graph showing the time course change of clobetasolpropionate concentration in aqueous humor after ophthalmicadministration of nanosuspension eye drop prepared in Examples 7(1) to7(4). The ordinate represents the clobetasol propionate concentration(ng/mL) in aqueous humor, and the abscissa represents the elapsed time(minutes) after the administration. The white circles indicate a 0.05%ophthalmic nanosuspension P (HPMC (60SH-50) 3 mg/mL), the black circlesindicate a 0.05% ophthalmic nanosuspension Q (HPMC (60SH-4000) 1.5mg/mL), the white triangles indicate a 0.05% ophthalmic nanosuspension R(MC (SM-100) 2 mg/mL), and the black triangles indicate a 0.05%ophthalmic nanosuspension S (MC (SM-4000) 1.5 mg/mL). The indicatedvalues are average, and the error bars indicate standard deviations.

FIG. 4 is a graph showing the time course change in clobetasolpropionate concentration in conjunctiva after ophthalmic administrationof nanosuspension eye drop prepared in Examples 7(1) to 7(4). Theordinate represents the clobetasol propionate concentration (ng/mL) inconjunctiva, and the abscissa represents the elapsed time (minutes)after the administration. The white circles indicate a 0.05% ophthalmicnanosuspension P (HPMC (60SH-50) 3 mg/mL), the black circles indicate a0.05% ophthalmic nanosuspension Q (HPMC (60SH-4000) 1.5 mg/mL), thewhite triangles indicate a 0.05% ophthalmic nanosuspension R (MC(SM-100) 2 mg/mL), and the black triangles indicate a 0.05% ophthalmicnanosuspension S (MC (SM-4000) 1.5 mg/mL). The indicated values areaverage, and the error bars indicate standard deviations.

FIG. 5 is a graph showing the inflammation score of the external eye fora rabbit model of BSA-induced uveitis. The ordinate represents theinflammation score, and the abscissa represents the elapsed days (fromthe 15th to 18th day) after the first BSA administration. The white barsindicate a control group (physiological saline solution), the dark graybars indicate a 0.05% clobetasol propionate ophthalmic nanosuspensionadministration group, and the pale gray bars indicate a positive controlgroup (0.1% fluorometholone ophthalmic solution administration group).The indicated values are average, and the error bars indicate standarddeviations.

FIG. 6 is a graph showing the inflammation score of the internal eye fora rabbit model of BSA-induced uveitis. The ordinate represents theinflammation score, and the abscissa represents the elapsed days (fromthe 15th to 18th day) after the first BSA administration. The white barsindicate a control group (physiological saline solution), the dark graybars indicate a 0.05% clobetasol propionate ophthalmic nanosuspensionadministration group, and the pale gray bars indicate a positive controlgroup (0.1% fluorometholone ophthalmic solution administration group).The indicated values are average, and the error bars indicate standarddeviations.

FIG. 7 presents graphs showing the inflammation scores of the externaleye (A) and internal eye (B) on the 29th day after the first BSAadministration for a rabbit model of BSA-induced uveitis. The ordinatesrepresent the inflammation scores. The white bars indicate a controlgroup (physiological saline solution), the dark gray bars indicate a0.05% clobetasol propionate ophthalmic nanosuspension administrationgroup, and the pale gray bars indicate a positive control group (0.1%fluorometholone ophthalmic solution administration group). The indicatedvalues are average, and the error bars indicate standard deviations.

FIG. 8 is a graph showing the conjunctival weight for a rat model ofcroton-induced conjunctivitis. The ordinate represents the conjunctivalweight (g). The indicated values are average, and the error barsindicate standard deviations.

FIG. 9 is a graph showing the palpebral conjunctival weight for a ratmodel of carrageenan-induced conjunctival edema. The ordinate representsthe palpebral conjunctival weight (g). The indicated values are average,and the error bars indicate standard deviations.

FIG. 10 is a graph showing the PGE2 concentration in aqueous humor for arabbit model of LPS-induced uveitis. The ordinate represents the PGE2concentration (pg/mL) in aqueous humor. The indicated values areaverage, and the error bars indicate standard deviations.

FIG. 11 is a graph showing the PGE2 concentration in vitreous body for arabbit model of LPS-induced uveitis. The ordinate represents the PGE2concentration (pg/mL) in vitreous body. The indicated values areaverage, and the error bars indicate standard deviations.

FIG. 12 is a graph showing the protein concentration in aqueous humorfor a rabbit model of puncture-induced anterior chamber inflammation.The ordinate represents the protein concentration (mg/mL) in anteriorchamber aqueous humor. The indicated values are average, and the errorbars indicate standard deviations.

FIG. 13 is a graph showing the PGE2 concentration in vitreous body for arabbit model of LPS-induced uveitis. The ordinate represents the PGE2concentration (pg/mL) in vitreous body. The indicated values areaverage, “b.i.d” means twice administration per day, and “q.i.d” meansfour-times administration per day.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

1. Aqueous Suspension Containing Nanoparticles of a GlucocorticosteroidCompound

The nanoparticle of a glucocorticosteroid compound can be produced bymixing the glucocorticosteroid compound with a physiologicallyacceptable salt and a physiologically acceptable polyol, and wetpulverizing the organic compound. The production method is described indetail in International Publication No. WO 2008/126797. The mixing steponly requires that glucocorticosteroid compound, the physiologicallyacceptable salt and the physiologically acceptable polyol are all mixedtogether in the end, and an order of addition is not limited. The mixingstep may be achieved by, for example, adding the physiologicallyacceptable salt and the physiologically acceptable polyol to theglucocorticosteroid compound or alternatively by adding theglucocorticosteroid compound to the physiologically acceptable salt andthe physiologically acceptable polyol. The glucocorticosteroid compoundnanoparticles contained in a powder of the present invention can beproduced by adding the physiologically acceptable salt and thephysiologically acceptable polyol to an organic compound having amelting point of 80° C. or more, and wet pulverizing the organiccompound. In this method, the aqueous suspension can be prepared withoutremoving the salt and the polyol. Since there is no need to remove thesalt and the polyol, the suspension can be prepared by very simplesteps. The wet pulverization is achieved by mixing the organic compound,the salt and the polyol, and kneading the mixture. The nanoparticle of aglucocorticosteroid compound can be preferably produced by addinglecithin during or after the pulverization step.

The glucocorticosteroid compound nanoparticle is produced preferably bywet pulverization without using a hard solid pulverization aid, morepreferably without using a solid pulverization aid such as glassproducts, metallic products such as stainless steel, ceramic productssuch as zirconia and alumina, and large molecular products such as rigidpolystyrene. Most preferably, the glucocorticosteroid compoundnanoparticle is produced by wet pulverization without using a solidpulverization aid other than the physiologically acceptable salt and theviscosity modifier.

The “physiologically acceptable” means it is believed not to causephysiological problems being administered into a body. The physiologicalacceptance of a certain substance is suitably determined depending onthe species to be administered the substance as well as modes ofadministration. Examples of the physiologically acceptable solventinclude substances which are approved as additives or solvents forpharmaceutical drugs or for food products.

The “physiologically acceptable salt” herein is not limited as long asit can be administered without causing physiological problems. Thephysiologically acceptable salt preferably has low solubility topolyols, high solubility to water and/or low moisture absorption withsuitable hardness for pulverization of the organic compound. Morepreferably, the physiologically acceptable salt used in the method forproducing the glucocorticosteroid compound nanoparticle has two or moreof these properties. The solubility of physiologically acceptable saltto polyols is preferably 10% (mass/volume) or less. The physiologicallyacceptable salt is preferably highly soluble to water for easy removalafter pulverization. Specific examples include the salts listed above inthis specification.

The “physiologically acceptable salt” is preferably pulverized foradjusting the particle diameter before mixing with theglucocorticosteroid compound. Moreover, the physiologically acceptablesalt may be dried under reduced pressure at a temperature of 30 to 200°C. to reduce a contained water for preventing particle fusion and growthcaused by the contained water. When the particle diameter of thephysiologically acceptable salt is adjusted in advance, the particlevolume mean diameter may be, for example, 5 to 300 μm, 10 to 200 μm,preferably 0.01 to 300 μm, more preferably 0.1 to 100 μm, furtherpreferably 0.5 to 50 μm, most preferably 1 to 5 μm. The amount of saltto be used is preferably 1 to 100 times, more preferably 5 to 30 times,further preferably 10 to 20 times a mass of the glucocorticosteroidcompound. One kind of salt may be used singly, or two or more kinds ofsalts may be used together.

The “physiologically acceptable polyol” used in the method for producingthe glucocorticosteroid compound nanoparticles is not limited as long asit can be administered without causing any physiological problems. Thepreferable physiologically acceptable polyol has low solubility tosalts, high solubility to water, a low freezing point and/or a highignition point. The physiologically acceptable polyol is preferablyhighly soluble to water for easy removal after pulverization.

The polyol used in the method for producing the glucocorticosteroidcompound nanoparticles preferably has a high viscosity. The viscosity ofa polyol at 20° C. may be 40 mPa·s or more, preferably 50 mPa·s or more,more preferably 80 mPa·s or more. The upper limit of the viscosity at20° C. of the polyol to be used in the method for producing theglucocorticosteroid compound nanoparticles is not limited, and, forexample, can be selected from the ranges from 40 mPa·s or more to 5,000mPa·s or less, preferably 50 mPa·s or more to 3,000 mPa·s or less, morepreferably 80 mPa·s or more to 2,000 mPa·s or less. Specific examplesinclude the polyols listed above in this specification.

The amount of physiologically acceptable polyol used in the method forproducing the glucocorticosteroid compound nanoparticles is preferably0.5 to 100 times, more preferably 1 to 10 times a mass of the organiccompound to be pulverized. The kind of polyol can be suitably determinedaccording to the solubility of the organic compound to be pulverized.One kind of polyol may be used singly, or two or more kinds of polyolsmay be used together.

In the method for producing nanoparticles of a glucocorticosteroidcompound, the kneaded product containing the glucocorticosteroidcompound, the polyol and the salt preferably has a high viscosity. Theviscosity of the kneaded product can be increased preferably by usingthe mixture in which a viscosity modifier is added to the polyol, or byadding a viscosity modifier independently from the polyol, which caneffectively improve the pulverization efficiency. The viscositymodifiers described in the above can be added to the polyol. Theviscosity at 20° C. of a polyol to which the viscosity modifier is addedis preferably 1,000 mPa·s or more, more preferably 2,000 mPa·s or more,further preferably 5,000 mPa·s or more, most preferably 10,000 mPa·s ormore. The upper limit of the viscosity at 20° C. of the polyol to whichthe viscosity modifier is added is not limited, and, for example, can beselected from the ranges from 1,000 mPa·s or more to 5,000,000 mPa·s orless, preferably 1,000 mPa·s or more to 1,000,000 mPa·s or less, morepreferably 2,000 mPa·s or more to 500,000 mPa·s or less, furtherpreferably 5,000 mPa·s or more to 300,000 mPa·s or less, most preferably10,000 mPa·s or more to 100,000 mPa·s or less.

In the method for producing the glucocorticosteroid compoundnanoparticles, any grinder can be used for wet pulverization of theglucocorticosteroid compound without limitation, as long as it canmechanically knead and disperse the glucocorticosteroid compound, thesalt, the polyol and/or the dispersion stabilizer. Examples of thecommonly used grinder include kneaders, two rolls, three rolls, fretmills, hoover mullers, and disk blade kneader dispersers.

The pulverization temperature can be suitably determined according tothe glucocorticosteroid compound to be pulverized and a type of grinder.The pulverization temperature is not limited, and preferably −50 to 50°C., more preferably −20 to 30° C., and most preferably −10 to 25° C. Thepulverization time can be also suitably determined according to theorganic compound to be pulverized and a type of grinder. Thepulverization time can be, for example, 1 to 50 hours, 2 to 30 hours, 3to 20 hours, 4 to 18 hours, 5 to 10 hours.

After completion of the pulverization of glucocorticosteroid compound,the objective particles of pulverized glucocorticosteroid compound canbe obtained without removing the salt and the polyol which are used forthe pulverization. Because washing step is not necessary, nanoparticlepreparation is produced simpler at a lower cost. The suspension can beprepared by homogenizing the mixture of the glucocorticosteroidcompound, the salt, the polyol and/or the viscosity modifier in asolvent using a homogenizer. The solvent used for homogenizing themixture is not limited as long as easily dissolving the polyol, the saltand the viscosity modifier but hardly dissolving the pulverizedglucocorticosteroid compound and being physiologically acceptable. Thesolvent is preferably water but any solvent other than water can beused, which includes a mixture of water and an organic solvent such asacetic acid, methanol or ethanol. The homogenized mixture can befiltered, if necessary. The filtration method is not limited and anyknown filtration method used for filtering organic compounds containedtherein can be employed. Examples of the filtration method include areduced pressure filtration method, an applied pressure filtrationmethod, and an ultrafiltration membrane method.

The pulverized particles typically have a high surface energy and thuseasily agglomerate. Thus, the agglomeration inhibitor described abovemay be added after removing the salts etc. to prevent the secondaryagglomeration. One kind of the agglomeration inhibitor may be usedsingly, or two or more kinds of agglomeration inhibitors may be usedtogether.

After removing the salt and the polyol, the obtained particles ofpulverized glucocorticosteroid compound can be dried to remove thesolvent used for removing the salt etc. The drying method is not limitedand any method commonly used for drying an organic compound can beemployed. Examples of the drying method include a reduced pressuredrying method, a freeze-drying method, a spray drying method, and aspray-freezing-drying method. The drying temperature and drying time inthese drying methods are not limited but is preferably at a lowtemperature for maintaining the chemical stability of organic compoundparticles for medical use and preventing the secondary particleagglomeration. By the same reason, freeze-drying method, the spraydrying method, and the spray-freezing-drying method are preferable.

The mean particle diameter ranges of the pulverized glucocorticosteroidcompound particles obtained by the above production method can be thesame as the mean particle diameter of the glucocorticosteroid compoundnanoparticles contained in the aqueous suspension or aqueouspharmaceutical composition described above. Also, the ranges of the 90%diameter (D90) and the 50% diameter (D50) of the pulverizedglucocorticosteroid compound particles obtained by the above productionmethod can be the same as the 90% diameter (D90) and the 50% diameter(D50), respectively, of the glucocorticosteroid compound nanoparticlescontained in the aqueous suspension or aqueous pharmaceuticalcomposition described above.

The “mean particle diameter” or “Dv” herein means the arithmetic meandiameter of the particle size distribution measured by dynamic lightscattering photon correlation spectroscopy. The 50% diameter (alsoreferred to as median diameter, D50) represents the diameter at whichpowder particles are divided into two groups in the particle sizedistribution measured by the above measurement method, wherein theamounts of particles are equal between said two groups, the largerdiameter group and the smaller diameter group. The “90% diameter” meansthe diameter (D90) of the particle at 90% position in the particle sizedistribution measured by the above measurement method, wherein thenumber of particles is counted from the smaller particle diameter to thelarger particle diameter, as setting 0% (less smallest) to 100% (thelargest particle). The “10% diameter” means the diameter (D10) of theparticle at 10% position in the particle size distribution measured bythe above measurement method, wherein the number of particles is countedfrom the smaller particle diameter to the larger particle diameter, assetting 0% (less smallest) to 100% (the largest particle). Themeasurement method by dynamic light scattering photon correlationspectroscopy, and the calculation method of particle size distributionare well known in the art.

2. Pharmaceutical Composition

The present invention relates to the pharmaceutical compositioncontaining nanoparticles of a glucocorticosteroid compound. Thepharmaceutical composition is preferably a pharmaceutical compositionfor parenteral administrations such as injections or topicalpreparations. The type of the pharmaceutical composition herein is notlimited. Examples of the formulation include topical eye preparations(e.g., eye drops), topical ear preparations (e.g., ear drops), topicalnose preparations (e.g., nose drops), suspensions, ointments, creams,gels, inhalers, injections (e.g., injections for intravenous injection,subcutaneous administration, intramuscular injection and intravenousdrips). These preparations can be produced in accordance with aconventional method. The pharmaceutical composition preferably containsa dispersion stabilizer. In case of an injection, the formulation may beprepared using the glucocorticosteroid compound nanoparticles suspendedin water, and may be suspended in saline or a glucose solution asnecessary, which may further be added a dispersant, a buffer or apreservative. The pharmaceutical composition can be formulated as aparenteral administration form including injection for intravenousadministration, intramuscular administration or subcutaneousadministration, intravenous drip, transdermal absorber, transmucosalabsorber, eye drop, ear drop, nose drop or inhaler.

The pharmaceutical composition may contain a pharmacologicallyacceptable carrier (an additive for preparations). The kind of additivesfor preparations used for manufacturing the pharmaceutical composition,the proportion of the additives for preparations relative to the activecomponent, or the method for manufacturing the pharmaceuticalcomposition may be suitably selected by the person skilled in the artdepending on the composition form. The additive for preparations can bean inorganic or organic, or a solid or liquid substance, and can betypically added in a range from 1 wt % to 90 wt % of the activecomponent weight. Specific examples of such substance include lactose,glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesiumaluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethyl cellulose calcium, ionexchange resin, methyl cellulose, gelatin, gum arabic,hydroxypropylcellulose, hydroxypropyl methylcellulose,polyvinylpyrrolidone, PVA, light anhydrous silicic acid, magnesiumstearate, talc, tragacanth, bentonite, veegum, titanium oxide, fattyacid sorbitan ester, sodium lauryl sulfate, glycerin, fatty acidglycerin ester, purified lanolin, glycerogelatin, polysorbate, Macrogol,vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon,nonionic surfactant, propylene glycol, water, benzalkonium chloride,hydrochloric acid, sodium chloride, sodium hydroxide, lactic acid,sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate,citric acid, sodium citrate, disodium edetate, Poloxamer 407 andpolycarbophil. For example, the pharmaceutical composition may containone or more additives for preparations selected from POE-POP glycol,PVA, hydroxypropyl methylcellulose and methyl cellulose.

The aqueous suspension or pharmaceutical composition can be in the formof a kit, accompanying an outer package, a container, a diluent, asuspension, and/or an instruction for a preparation/administration. Whenthe aqueous suspension or pharmaceutical composition is provided in theform of a kit, different components of the aqueous suspension orpharmaceutical composition may be individually packed in separatecontainers and contained in a single kit. Alternatively, more than onebut not all of the components of the aqueous suspension orpharmaceutical composition may be included in the kit (at least theglucocorticosteroid compound nanoparticles is included in the kit), andother components may be provided separately from the kit. When theaqueous suspension or pharmaceutical composition is provided in the formof a kit, the necessary components are preferably mixed immediatelybefore use to obtain the aqueous suspension or pharmaceuticalcomposition of the present invention.

The kit of the present invention, for example, can be as follows:

(a) a kit for preparing a pharmaceutical composition comprising anaqueous suspension containing nanoparticles of a glucocorticosteroidcompound;

(b) the kit of (a) further comprising a dispersion stabilizer;

(c) the kit of (b), wherein the dispersion stabilizer is one or moresubstances selected from POE-POP glycol, PVA, hydroxypropylmethylcellulose, and methyl cellulose;

(d) the kit of any one of (a) to (c), for preparing a pharmaceuticalcomposition for parenteral administration;

(e) the kit of any one of (a) to (d), for preparing an injection or atopical preparation;

(f) the kit of (e), for preparing a topical eye preparation, a topicalear preparation, a topical nose preparation or a topical lungpreparation, or an eye drop, an ear drop, a nose drop or an inhaler;

(g) The kit of any one of (a) to (f), wherein the pharmaceuticalcomposition is a therapeutic drug or a preventive drug for aninflammatory or infectious disease of the eye, ear, nose or lung.

In one embodiment, the present invention may be the method for preparingan aqueous pharmaceutical composition containing the glucocorticosteroidcompound nanoparticles, comprising mixing a diluent and the aqueoussuspension containing the glucocorticosteroid compound nanoparticles.

In preparing the pharmaceutical composition (e.g., injections, topicaleye preparations (preferably eye drops), topical ear preparations(preferably ear drops), topical nose preparations (preferably nosedrops) or topical lung preparations (preferably inhalers)), the pH andosmotic pressure are not limited as long as they are acceptable for thetopical preparations, and preferably is pH 5 to 9.5, more preferably ispH 6 to 9, further preferably is pH 7 to 9. The ratio of osmoticpressure of the preparation (except ointments) to saline is, forexample, about 0.3 to 4.3, preferably about 0.3 to 2.2, particularlypreferably about 0.5 to 1.5. The pH and osmotic pressure can becontrolled using a pH control agent, a tonicity agent or salts by amethod known in the art.

The pharmaceutical composition can be suitably produced by a knownmethod, for example, by mixing the aqueous suspension containing theglucocorticosteroid compound nanoparticles with desired components in asuitable diluent such as distilled water or purified water, adjustingthe above osmotic pressure and pH, subjecting to high pressure steamsterilization or filter-sterilization under aseptic conditions, andfilling aseptically in a washed sterilized container.

The pharmaceutical composition can be a therapeutic or preventive agentfor inflammatory or infectious diseases. For example, the pharmaceuticalcomposition can be a therapeutic or preventive agent for inflammatory orinfectious diseases caused by infections. The present inventionencompasses the aqueous suspension containing the glucocorticosteroidcompound nanoparticles and a dispersion stabilizer for the use as apharmaceutical (a therapeutic or preventive drug for inflammatory orinfectious diseases).

The inflammatory or infectious disease herein encompasses systemicinflammatory and infectious diseases and topical inflammatory andinfectious diseases. The inflammatory diseases include, in addition tothe inflammatory diseases caused by infections, allergic inflammatorydiseases (e.g., allergic rhinitis, allergic conjunctivitis, allergicdermatitis, allergic eczema, allergic asthma and allergic pneumonia).Examples of the systemic inflammatory disease include systemicinflammatory or infectious diseases such as superficial/deep skininfections, lymphangitis/lymphadenitis, mastitis, osteomyelitis,tonsillitis, pneumonia, pyelonephritis, urethritis, gonococcalinfection, syphilis, intrauterine infection, scarlet fever, diphtheria,whooping cough, secondary infections from external wounds/burns andsurgeries, pharyngitis/laryngitis, bronchitis, secondary infections fromchronic respiratory diseases, pericoronitis, periodontal inflammation,tetanus, cystitis, prostatitis, infectious enteritis, jaw inflammation,infectious arthritis and gastritis.

The pharmaceutical composition can be specifically used for treating orpreventing eye inflammatory and infectious diseases and various symptomsassociated therewith. Examples of the eye inflammatory and infectiousdisease include eye lid symptoms such as blepharitis,blepharoconjunctivitis, meibomitis, acute or chronic stye, chalazion,dacryocystitis, dacryoadenitis and acne rosacea; conjunctival symptomssuch as conjunctivitis, ophthalmia neonatorum and trachoma; cornealsymptoms such as corneal ulcer, superficial keratitis and interstitialkeratitis, ketatoconjunctivitis, foreign objects and post-surgeryinfections; and anterior chamber and uvea symptoms such asendophthalmitis, infectious uveitis and post-surgery infections. Theprevention of infections includes the administration before surgicaltreatment such as an operation or before contacting a person presentinginfectious symptoms. In using for the prevention, an administration canbe before surgical treatments such as blepharoplasty, chalazion removal,blepharorrhaphy, surgeries for canaliculi and lacrimal drainage systemand other surgical treatments relating to eyelids and lacrimalapparatus; conjunctival surgeries such as removal of pterygium,pinguecula or tumors, conjunctival transplant, external wounds such ascuts, burns and scratches and conjunctival flap surgery; cornealsurgeries such as removal of foreign objects, keratotomy and cornealtransplant; refractive surgeries such as photorefractive procedure;glaucoma surgeries such as bleb filtration; anterior chamberparacentesis; iridotomy; cataract surgery; retinal surgery; andextraocular muscle relating surgeries. The prevention of ophthalmianeonatorum is also included in the prevention defined herein.

The pharmaceutical composition of the present invention, for example,can be used for treating or preventing various symptoms associated withinflammatory or infectious diseases of the ear. Examples of theinflammatory or infectious disease of the ear include otitis media andotitis externa. The prevention of infectious diseases includespresurgical treatments and treatments given before conditions ofpossible infections (e.g., contacts with a person infected or possiblyinfected). Examples of the prevention include an administration givenbefore surgical treatments associated with external wounds or damages ofthe ear and other surgeries or treatments.

The pharmaceutical composition further can treat or prevent varioussymptoms associated with inflammatory or infectious diseases of thenose. Throughout the entire specification, the term “nose” used in thephrases of “inflammatory or infectious diseases of the nose” and the“topical nose preparation” means the entire upper respiratory tract, forexample, the nasal cavity, nasopharynx, pharynx and larynx. Examples ofthe inflammatory or infectious disease of the nose include sinusitis,allergic rhinitis and rhinitis.

The pharmaceutical composition can further be used to treat or preventvarious symptoms associated with inflammatory or infectious diseases ofthe lung. Throughout the entire specification, the term “lung” used inthe phrases of “inflammatory or infectious diseases of the lung” and the“topical lung preparation” means the entire lower respiratory tract, forexample, the trachea, bronchus, bronchiole and lung. Examples of theinflammatory or infectious diseases of the lung include pneumonia,bronchitis, allergic pneumonia and asthma.

More preferably, the pharmaceutical composition can be used to treat orprevent infectious diseases (e.g., infectious diseases of the eye, ear,nose or lung) caused by various bacteria or parasite. Examples of suchmicroorganism include the Staphylococcus genus such as Staphylococcusaureus and Staphylococcus epidermidis; the Streptococcus genus such asStreptococcus pneumoniae and Streptococcus pyogenes, Groups C, F and GStreptococci and Group viridans Streptococcus; Haemophilus influenzaeincluding biotype III; Haemophilus ducreyi; Moraxella catarrhalis; theNeisseria genus such as Neisseria gonorrhoeae and Neisseriameningitidis; the Chlamydia genus such as Chlamydia trachomatis,Chlamydia psittaci and Chlamydia pneumoniae; the Mycobacterium genussuch as atypical mycobacteria including Mycobacterium tuberculosis andMycobacterium tubercule bacillus intracellular complex and Mycobacteriummarinum, Mycobacterium fortuitum and Mycobacterium chelonae; Bordetellapertussis; Campylobacter jejuni; Legionella pneumophila; Bacteroidesbivius; Welch bacillus; Peptostreptococcus species; Borreliaburgdorferi; Mycoplasma pneumonia; Treponema pallidum; Ureaplasmaurealyticum; Toxoplasma; Malaria; and nosema.

3. Treatment Method/Prevention Method

The pharmaceutical composition of the present invention can be used totreat or prevent inflammatory or infectious diseases by beingadministered in an effective amount to a patient in need thereof. Thepresent invention accordingly relates to a method for treatment orprevention of inflammatory or infectious diseases, comprisingadministering an effective amount of the pharmaceutical compositioncontaining the aqueous suspension containing the glucocorticosteroidcompound nanoparticles (and a dispersion stabilizer) to a patient inneed thereof. The patient herein includes any animals classified in themammals including but not limited to human; companion animals such asdogs, cats and rabbits; domestic animals such as cows, pigs, sheep andhorses, in which human is preferable.

The dose and number of administration of the pharmaceutical compositionare not limited and can be suitably selected at a physician's discretiondepending on purpose of prevention of deterioration/progress and/or thepurpose of treatment of the disease to be treated, the type of disease,and patient's conditions such as body weight and age. The dose isgenerally about 0.01 to 1000 mg (on an active component weight basis) aday for an adult, and can be administered once or in several times aday. The administration route is an injection or topical administration,for example, intravenous injections, intramuscular injections orsubcutaneous injections, intravenous drips, eye drops, ear drops, nosedrops, transdermal administration, transmucosal administration orinhalation. The content of the effective agent in the pharmaceuticalcomposition can be, for example, 0.001% to 10%, 0.01% to 1% or 0.05% to0.1%.

When the pharmaceutical composition of the present invention is in theform of an injection, it can be administered continuously orintermittently in a daily dose of 0.001 to 100 mg (on an activecomponent weight basis) for an adult.

When the aqueous pharmaceutical composition of the present invention isfor topical administration, it is directly administered to a topicalarea such as an affected site, an area around an affected site, or anorgan including an affected site. The pharmaceutical composition of thepresent invention, for example, can be formed into a topical eyepreparation, a topical ear preparation, a topical nose preparation or atopical lung preparation. When the pharmaceutical composition of thepresent invention is a topically administrable preparation, it isapplicable every day or in any number of times after a topicalinflammatory or infectious disease is developed. The application amountcan be suitably determined depending on conditions, and is typicallyapplied to the eye once to six times a day, for example, once, twice,three times, four times, five times or six times a day, with about 1 to3 drops per application. The administration period can be any period oftime until symptoms subside adequately, for example, two weeks to oneyear.

The present invention is illustrated in more detail below in examples,but which does not intend to limit the scope of the present invention.All of the documents cited throughout this application are incorporatedherein by reference in their entirety.

(Example 1) Study on Pulverization of Clobetasol Propionate

In order to examine an effect of anhydrous citric acid and hydrogenatedsoybean lecithin on pulverization of clobetasol propionate, thefollowing pulverizations (1) to (9) were conducted and the mean particlediameter (Dv), the median particle diameter (D50) and the 90% particlediameter (D90) of the obtained particles were measured using a particlesize distribution analyzer (DelsaNano S, Beckman Coulter, Inc.)

(1) Pulverization without Adding Anhydrous Citric Acid or HydrogenatedSoybean Lecithin

10 g of clobetasol propionate (melting point: 193 to 200° C., TokyoChemical Industry Co., Ltd.) having a mean particle diameter of 38,390nm and 110 g of sodium chloride (Tomita Salt K-30, Tomita PharmaceuticalCo., Ltd.) were charged into a 1.0 L water-cooling vertical kneader(INOUE MFG., INC.) and homogeneously mixed. To the mixture, 17 g ofglycerin (Sigma-Aldrich Co. LLC.) was added with keeping the mixture inform of dough, and pulverized at 5° C. for 6 hours. Subsequently, 0.1 gof the obtained pulverized-kneaded product (dough) and 5 g of 0.1%POE-POP glycol (UNILUB 70DP-950B, NOF CORPORATION) as the dispersantwere weighed into a 50 mL screw bottle, which was dispersedhomogeneously using a ultrasonic device (MODEL VS-100III, AS ONECorporation), and added 45 g of purified water to obtain 50 g of asuspension. The obtained suspension was measured for the particle sizedistributions using a particle size distribution analyzer (DelsaNano S,Beckman Coulter, Inc.). The particle size distributions of clobetasolpropionate were measured to have a mean particle diameter (Dv) of 285nm, a median particle diameter (D50) of 231 nm and the 90% particlediameter (D90) of 433 nm.

(2) Pulverization with Addition of Anhydrous Citric Acid

Except for adding 0.8 g of anhydrous citric acid (JUNSEI CHEMICAL CO.,LTD.) to the mixture, the pulverization was conducted in the same manneras Example 1 (1) at 5° C. for 7 hours. Subsequently, thepulverized-kneaded product (dough) was dispersed in the same manner asExample 1. The clobetasol propionate was measured for the particle sizedistributions, which showed a mean particle diameter (Dv) of 260 nm, amedian particle diameter (D50) of 222 nm and a 90% particle diameter(D90) of 363 nm.

(3) Pulverization with Addition of Hydrogenated Soybean Lecithin

Except for adding 10 g of hydrogenated soybean lecithin (Phospholipon90H, Lipoid GmbH) to the mixture, the pulverization and subsequentdispersion were conducted in the same manner as Example 1 (1). Theparticle size distributions of clobetasol propionate were measured tohave a mean particle diameter (Dv) of 147 nm, a median particle diameter(D50) of 124 nm and a 90% particle diameter (D90) of 210 nm.

(4) Pulverization with Addition of Anhydrous Citric Acid andHydrogenated Soybean Lecithin 1

Except for adding 0.8 g of anhydrous citric acid (JUNSEI CHEMICAL CO.,LTD.) and 5 g of hydrogenated soybean lecithin (Phospholipon 90H, LipoidGmbH) to the mixture, the pulverization and subsequent dispersion wereconducted in the same manner as Example 1 (1). The particle sizedistributions of clobetasol propionate were measured to have a meanparticle diameter (Dv) of 166 nm, a median particle diameter (D50) of138 nm and a 90% particle diameter (D90) of 241 nm.

(5) Pulverization with Addition of Anhydrous Citric Acid andHydrogenated Soybean Lecithin 2

Except for adding 0.8 g of anhydrous citric acid (JUNSEI CHEMICAL CO.,LTD.) and 10 g of hydrogenated soybean lecithin (Phospholipon 90H,Lipoid GmbH) to the mixture, the pulverization was conducted in the samemanner as Example 1 (1) at 5° C. for 7 hours. Subsequently, thepulverized-kneaded product (dough) was dispersed in the same manner asExample 1. The clobetasol propionate was measured for the particle sizedistributions, which showed a mean particle diameter (Dv) of 101 nm, amedian particle diameter (D50) of 87 nm and a 90% particle diameter(D90) of 141 nm.

(6) Pulverization with Addition of Anhydrous Citric Acid andHydrogenated Soybean Lecithin 3

Except for adding 0.8 g of anhydrous citric acid (JUNSEI CHEMICAL CO.,LTD.) and 20 g of hydrogenated soybean lecithin (Phospholipon 90H,Lipoid GmbH) to the mixture, the pulverization was conducted in the samemanner as Example 1 (1) at 5° C. for 7 hours. Subsequently, thepulverized-kneaded product (dough) was dispersed in the same manner asExample 1. The clobetasol propionate was measured for the particle sizedistributions, which showed a mean particle diameter (Dv) of 144 nm, amedian particle diameter (D50) of 121 nm and a 90% particle diameter(D90) of 214 nm.

(7) Pulverization with Addition of Anhydrous Citric Acid andHydrogenated Soybean Lecithin 4

Except for adding 2 g of anhydrous citric acid (JUNSEI CHEMICAL CO.,LTD.) and 5 g of hydrogenated soybean lecithin (Phospholipon 90H, LipoidGmbH) to the mixture, the pulverization was conducted in the same manneras Example 1 (1) at 5° C. for 7 hours. Subsequently, 0.1 g of theobtained pulverized-kneaded product (dough) and 5 g of 0.01% POE-POPglycol (UNILUB 70DP-950B, NOF CORPORATION) as the dispersant wereweighed into a 50 mL screw bottle, and dispersed homogeneously using aultrasonic device (MODEL VS-100III, AS ONE Corporation), to which 15 gof purified water was added to obtain 20 g of a suspension. The obtainedsuspension was measured for the particle size distributions using aparticle size distribution analyzer (DelsaNano S, Beckman Coulter,Inc.). The particle size distributions of clobetasol propionate werefound to have a mean particle diameter (Dv) of 137 nm, a median particlediameter (D50) of 112 nm and a 90% particle diameter (D90) of 209 nm.

(8) Pulverization with Addition of Anhydrous Citric Acid andHydrogenated Soybean Lecithin 5

Except for adding 2 g of anhydrous citric acid (JUNSEI CHEMICAL CO.,LTD.) and 10 g of hydrogenated soybean lecithin (Phospholipon 90H,Lipoid GmbH) to the mixture, the pulverization was conducted in the samemanner as Example 1 (1) at 5° C. for 6 hours. Subsequently, 0.1 g of theobtained pulverized-kneaded product (dough) was dispersed in the samemanner as Example 1 (1). The obtained suspension was measured for theparticle size distributions. The particle size distributions ofclobetasol propionate were found to have a mean particle diameter (Dv)of 129 nm, a median particle diameter (D50) of 112 nm and a 90% particlediameter (D90) of 179 nm.

(9) Pulverization with Addition of Anhydrous Citric Acid andHydrogenated Soybean Lecithin 6

Except for adding 2 g of anhydrous citric acid (JUNSEI CHEMICAL CO.,LTD.) and 20 g of hydrogenated soybean lecithin (Phospholipon 90H,Lipoid GmbH) to the mixture, the pulverization was conducted in the samemanner as in Example 1 (1) at 5° C. for 7 hours. Subsequently, 0.1 g ofthe obtained pulverized-kneaded product (dough) was dispersed in thesame manner as Example 1 (1). The obtained suspension was measured forthe particle size distributions. The particle size distributions ofclobetasol propionate were found to have a mean particle diameter (Dv)of 147 nm, a median particle diameter (D50) of 121 nm and a 90% particlediameter (D90) of 228 nm.

Table 1 shows the conditions of pulverization (1) to (9) and theparticle diameters obtained as the result of pulverizations. Thisresults suggest that the pulverization formulation (5) showed the bestpulverization performance.

TABLE 1 Particle size distribution Pulverization formulation (g)measured after Hydrogenated pulverization (nm) Pulverization SodiumAnhydrous soybean Mean method CP chloride citric acid lecithin Glycerin(Dv) D10 D50 D90 (1) 10 110 — — 17 285 135 231 433 (2) 10 110 0.8 — 17260 143 222 363 (3) 10 110 — 10 17 147 78 124 210 (4) 10 110 0.8 5 17166 86 138 241 (5) 10 110 0.8 10 17 101 56 87 141 (6) 10 110 0.8 20 17144 71 121 214 (7) 10 110 2 5 17 137 65 112 209 (8) 10 110 2 10 17 12970 112 179 (9) 10 110 2 20 17 147 66 121 228

(Example 2) Study on Formulation of Clobetasol Propionate

(1) Study on the Dispersant

0.1 g of the pulverized-kneaded product (dough) obtained in Example 1(4) and 5 g of an aqueous solution containing each dispersant shown inTable 2 were weighed into a 50 mL screw bottle, and dispersedhomogeneously using a ultrasonic device (MODEL VS-100III, AS ONECorporation), to which 45 g of purified water was added to obtain 50 gof a dispersion. Each of the obtained dispersions was stored at roomtemperature (about 25° C.) for 1 day. The transparency and the presenceof precipitation were visually observed immediately after the dispersionand after 1 day storage to evaluate the stability of dispersions.

The results are shown in Table 2. The symbols used in Table 2 todescribe the evaluated storage stability mean as follows. Good: goodstability, Fair: stable immediately after dispersion but precipitationwas generated as time advances; Poor: unstable, turbidity was identifiedimmediately after preparation. The test results shown in Table 2revealed that the suspension using POE-POP glycol (PLONON 407P, PluronicF68, UNILUB 70DP-950B) and PVA (Kuraray POVAL 217C) as the dispersantexhibited no precipitation detected and the transparency maintained withgood stability at both of immediately after dispersion and even after 1day storage.

TABLE 2 Dispersion state Immediately Storage Dispersant after stability(Concentration) General name Manufacturer dispersing After 1 dayevaluation HCO60 (0.1%) Polyoxyethylene Nikko Transparent Full Fairhydrogenated Chemicals precipitation castor oil 60 HCO40 (0.1%)Polyoxyethylene Nikko Transparent Full Fair hydrogenated Chemicalsprecipitation castor oil 40 Tween80 (0.1%) Polysorbate 80 NikkoTransparent Full Fair Chemicals precipitation Tween20 (0.1%) Polysorbate20 Sigma-Aldrich Transparent Full Fair precipitation MYS40MV (0.1%)Polyoxyl 40 Nikko Transparent Full Fair stearate Chemicals precipitationPLONON 407P Polyoxyethylene NOF Transparent Transparent Good (0.1%)polyoxypropylene CORPORATION glycol Pluronic F68 Polyoxyethylene NOFTransparent Transparent Good (0.1%) polyoxypropylene CORPORATION glycolUNILUB 70DP- Polyoxyethylene NOF Transparent Transparent Good 950B(0.1%) polyoxypropylene CORPORATION glycol Kuraray POVAL Polyvinylalcohol Kuraray Transparent Transparent Good 217C (0.1%) Chondroitinsulfate Sodium chondroitin Wako Pure Turbid Full Poor C sodium saltsulfate Chemical precipitation (0.1%) Industries PolyvinylpyrrolidonePolyvinylpyrrolidone Wako Pure Turbid Turbid Poor K-25 (0.1%) ChemicalIndustries Poly(ethylene Poly(ethylene KANTO Turbid Full Poor glycol)400 (0.1%) glycol) CHEMICAL precipitation Poly(ethylene Poly(ethyleneKANTO Turbid Full Poor glycol) 4000 (0.1%) glycol) CHEMICALprecipitation(2) Study on the Thickener

0.1 g of the pulverized-kneaded product (dough) obtained in Example 1(4) and 7.3 g of an aqueous solution of mixture of 0.1% PluronicF68/0.01% Tween80 (1:1) were weighed into a 50 mL screw bottle, anddispersed homogeneously for 3 minutes using a ultrasonic homogenizer(Sonicator S-4000, #418 microtip, power output 30, Astrason), to which1.5 g of the aqueous solution containing each of the thickeners shown inTable 3 was added and subsequently 13.5 g of purified water was added toobtain 22.4 g of a dispersion. The final concentration of each thickenerwas as shown in Table 3. Each of the obtained dispersions was stored atroom temperature (about 25° C.) for 4 days and the transparency and thepresence of precipitation was visually observed to evaluate thestability.

The results are shown in Table 3. The symbols used in Table 3 todescribe the evaluated storage stability mean as follows. Good: goodstability; Fair: stability is low, modest precipitation was found; Poor:unstable, full precipitation was observed. The test results shown inTable 3 revealed that the suspension using hydroxypropyl methylcelluloseand methylcellulose as the thickener exhibited no precipitation detectedand the transparency maintained with good stability at both ofimmediately after dispersion and even after 4 days storage.

TABLE 3 Visual Final observation Storage concentration (after 4stability Thickener General name Manufacturer (%) days) evaluationPurified water (no — — 0 Full Poor addition of precipitation thickener)TC-5 (R) Hydroxypropyl Shin-Etsu 0.1 Transparent, Good methylcelluloseChemical with no precipitation Metlose 60SH-50 Hydroxypropyl Shin-Etsu0.1 Transparent, Good methylcellulose Chemical with no precipitationKuraray POVAL Polyvinyl alcohol Kuraray 0.1 Transparent, Fair 217C withmodest precipitation Chondroitin sulfate Chondroitin sulfate Wako Pure0.1 Full Poor C sodium salt Chemical precipitation IndustriesPolyvinylpyrrolidone Polyvinylpyrrolidone Wako Pure 0.05 Full Poor K-90Chemical precipitation Industries Poly(ethylene Poly(ethylene KANTO 0.1Full Poor glycol) 6000 glycol) CHEMICAL precipitation Metlose SM-100Methyl cellulose Shin-Etsu 0.1 Transparent, Good Chemical with noprecipitation Metlose SM-15 Methyl cellulose Shin-Etsu 0.1 Transparent,Good Chemical with no precipitation HEC Hydroxyethyl Wako Pure 0.1 FullPoor cellulose Chemical precipitation Industries HIVISWAKO 104Carboxyvinyl Wako Pure 0.1 Full Poor polymer Chemical precipitationIndustries(3) Study on the Preservative 1

0.1 g of the pulverized-kneaded product (dough) obtained in Example 1(4), 7.3 g of an aqueous solution of mixture of 0.1% Pluronic F68/0.01%Tween80 (1:1), and 1.43 g of a 1% Kurary POVAL 217C aqueous solutionwere weighed into a 50 mL screw bottle, and dispersed homogeneously for7 minutes using a ultrasonic homogenizer (Sonicator S-4000, #418microtip, power output 30, Astrason), to which 1.43 g of a 0.01%benzalkonium chloride aqueous solution and 1.43 g of a 3% TC-5® aqueoussolution were added. To thus obtained mixture, a 100 mM sodium citrateaqueous solution was gradually added with stirring up to pH 7.0, towhich purified water was added to obtain 14.6 g of an eye drop. Theobtained eye drop was stored at a cycle of 5° C.-25° C. or at 40° C. for7 days and the transparency was visually observed to evaluate thestability.

The results of Example 2 (3) are shown in Table 4. The “cycle (5° C.-25°C.)” in the storage temperature in Table 4 means that the eye drop wasstored repeatedly at 5° C. for 6 hours and then at 25° C. for 6 hours.The test results shown in Table 4 revealed that the eye drop preparedusing benzalkonium chloride as the preservative maintained thetransparency and had good stability at both of immediately afterpreparation and even after 7 days storage.

TABLE 4 Stored Storage temperature duration (days) Cycle (5° C.-25° C.)40° C. 0 Transparent Transparent 7 Transparent Transparent

(Example 3) Study on the Filter-Sterilization

(1) Preparation of an Eye Drop 1

6.0 g of the pulverized-kneaded product (dough) obtained in Example 1(5), 408 g of a 0.01% UNILUB 70DP-950B aqueous solution, and 81.6 g of a1.0% Kurary POVAL 217C aqueous solution were added to a 1 L-beaker,roughly dispersed using a ultrasonic device (MODEL VS-100III, AS ONECorporation), and then uniformly dispersed using a high pressurehomogenizer (L01-YH1, 90 MPa×5 passes, SANWA ENGINEERING LTD.). To theobtained mixture, 7.48 g of a 0.1% benzalkonium chloride aqueoussolution and 7.48 g of a 3% TC-5® aqueous solution were added, which wasstirred for 5 minutes. A 100 mM sodium citrate aqueous solution wasadded thereto up to pH 7.0, to which purified water was added withstirring to give the total amount of 748 g. The obtained eye drop wasmeasured for the particle size distributions using a particle sizedistribution analyzer (DelsaNano S, Beckman Coulter, Inc.), which showedto have a mean particle diameter (Dv) of 173 nm, a median particlediameter (D50) of 151 nm and a 90% particle diameter (D90) of 233 nm.

(2) Preparation of an Eye Drop 2

6.0 g of the pulverized-kneaded product (dough) obtained in Example 1(7), 414 g of a 0.01% UNILUB 70DP-950B aqueous solution, and 82.8 g of a1.0% Kurary POVAL 217C aqueous solution were added to a 1 L-beaker,roughly dispersed using a ultrasonic device (MODEL VS-100III, AS ONECorporation), and then uniformly dispersed using a high pressurehomogenizer (L01-YH1, 90 MPa×5 passes, SANWA ENGINEERING LTD.). To theobtained mixture, 7.5 g of a 0.1% benzalkonium chloride aqueous solutionand 7.5 g of a 3% TC-5® aqueous solution were added, and stirred for 5minutes. A 100 mM sodium citrate aqueous solution was added thereto upto pH 7.0, to which purified water was added with stirring to give atotal amount of 750 g. The obtained eye drop was measured for theparticle size distributions using a particle size distribution analyzer(DelsaNano S, Beckman Coulter, Inc.) which showed a mean particlediameter (Dv) of 201 nm, a median particle diameter (D50) of 177 nm anda 90% particle diameter (D90) of 260 nm.

(3) Preparation of an Eye Drop 3

To a 1 L-beaker, 6.29 g of the pulverized-kneaded product (dough)obtained in Example 1 (8), 415 g of a 0.01% UNILUB 70DP-950B aqueoussolution, and 83.0 g of a 1.0% Kurary POVAL 217C aqueous solution wereadded, and roughly dispersed using a ultrasonic device (MODEL VS-100111,AS ONE Corporation), which was then uniformly dispersed using a highpressure homogenizer (L01-YH1, 90 MPa×5 passes, SANWA ENGINEERING LTD.).To the mixture, 7.84 g of a 0.1% benzalkonium chloride aqueous solutionand 7.84 g of a 3% TC-5® aqueous solution were added, and the mixturewas stirred for 5 minutes. The pH of the mixture was adjusted with 100mM sodium citrate aqueous solution up to pH 7.0. Then purified water wasadded with stirring to give the total amount of 784 g. The obtained eyedrop was measured for the particle size distributions using a particlesize distribution analyzer (DelsaNano S, Beckman Coulter, Inc.), whichshowed a mean particle diameter (Dv) of 204 nm, a median particlediameter (D50) of 166 nm and a 90% particle diameter (D90) of 306 nm.

(4) Study on the Filtration Permeability

Each of the eye drops prepared in Examples 3 (1) to (3) was tested forthe filtration permeability using two types of filter membranes(Optiscale 25 and Optiscale 25 Capsule) manufactured by Merck MilliporeCorporation. Filtration conditions were as follows.

Filter Names:

Optiscale 25 (pre-filter 0.5 μm/main filter 0.22 μm)

Optiscale 25 Capsule (pre-filter 0.2 μm/main filter 0.22 μm)

Filter material: polyvinylidene fluoride (PVDF)

Effective filtration area: 3.5 cm²

Test pressure: 0.18 MPa

The test was conducted by the Vmax method which measures a permeationflow rate of the eye drop over time to estimate the maximum processingamount of the filter, and the filtration was not continued until thefilter completely clogs.

The results are shown in Table 5. The permeation amount shown in Table 5represents the converted value of permeated amount of each eye dropthrough the filter to L/m². The permeation ratio is a percentage of thepost-filtration concentration relative to the pre-filtrationconcentration, in which the pre- and post-filtration concentrations ofclobetasol propionate were measured by HPLC. The results shown in Table5 revealed that all of the particle diameters can be sterilized byfiltration. The eye drop prepared in Example 3 (1) containing thesmallest particle diameter of the clobetasol propionate afterpulverization showed the highest values in both permeation amount andpermeation ratio.

TABLE 5 Filtration Filter name Drug (Pore size: Permeation PermeationEye drop concentration Pre-filter/ amount ratio sample (%) main filter)(L/m²) (%) (1) 0.05 Optiscale25 644 95.5 (0.5/0.22) Optiscale25 501 93.0Capsule (0.2/0.22) (2) 0.05 Optiscale25 450 70.8 (0.5/0.22) Optiscale25659 72.2 Capsule (0.2/0.22) (3) 0.05 Optiscale25 259 87.4 (0.5/0.22)Optiscale25 347 89.1 Capsule (0.2/0.22)

(Example 4) Pulverization of Clobetasol Propionate

(1) Production of Nanoparticles with a Mean Particle Diameter of 100 to150 nm

To a 1.0 L water-cooling vertical kneader (INOUE MFG., INC.), 10 g ofclobetasol propionate (melting point: 193 to 200° C., Tokyo ChemicalIndustry Co., Ltd.) having a mean particle diameter of 38,390 nm, 110 gof sodium chloride (Tomita Salt K-30, Tomita Pharmaceutical Co., Ltd.),10 g of hydrogenated soybean lecithin (Phospholipon 90H, Lipoid GmbH)and 0.8 g of anhydrous citric acid (JUNSEI CHEMICAL CO., LTD.) werecharged, and homogeneously mixed. To the mixture, 17 g of glycerin(Sigma-Aldrich Co. LLC.) was added with keeping the mixture in a stateof dough, and pulverized at 5° C. for 7 hours. Subsequently, 0.1 g ofthe obtained pulverized-kneaded product (dough) and 5 g of 0.01% POE-POPglycol (UNILUB 70DP-950B, NOF CORPORATION) as the dispersant wereweighed into a 50 mL screw bottle, which were dispersed homogeneouslyusing a ultrasonic device (MODEL VS-100III, AS ONE Corporation), and 45g of purified water was added thereto to obtain 50 g of a suspension.The obtained suspension was measured for the particle size distributionsusing a particle size distribution analyzer (DelsaNano S, BeckmanCoulter, Inc.), and the particle size distributions of clobetasolpropionate were found to have a mean particle diameter (Dv) of 101 nm, a10% particle diameter (D10) of 56 nm, a median particle diameter (D50)of 87 nm and a 90% particle diameter (D90) of 141 nm.

(2) Production of Nanoparticles with a Mean Particle Diameter of 100 to150 nm

Clobetasol propionate was pulverized and measured for the particle sizedistributions in the same manner as (1). The particle size distributionsof clobetasol propionate were found to have a mean particle diameter(Dv) of 108 nm, a 10% particle diameter (D10) of 57 nm, a medianparticle diameter (D50) of 89 nm and a 90% particle diameter (D90) of151 nm.

(3) Production of Nanoparticles with a Mean Particle Diameter of 250 to300 nm

Clobetasol propionate was pulverized and measured for the particle sizedistributions in the same manner as (1), except for no addition of 10 gof hydrogenated soybean lecithin (Phospholipon 90H, Lipoid GmbH). Theparticle size distributions of clobetasol propionate were found to havea mean particle diameter (Dv) of 260 nm, a 10% particle diameter (D10)of 143 nm, a median particle diameter (D50) of 222 nm and a 90% particlediameter (D90) of 363 nm.

(4) Production of Nanoparticles with a Mean Particle Diameter of 500 to700 nm

Into the Mortar Grinder RM 200 (Retsch GmbH), 1 g of clobetasolpropionate having a mean particle diameter of 38,390 nm and 2 g of amixture of sodium chloride and glycerin (sodium chloride 11 g, glycerin2 g) were charged and pulverized repeatedly nine times for 1 minute peroperation at room temperature. Subsequently, 0.04 g of the obtainedpulverized-kneaded product (dough) and 5 g of 0.01% POE-POP glycol(UNILUB 70DP-950B) as the dispersant were weighed into a 50 mL screwbottle, which was dispersed homogeneously using a ultrasonic device. Tothe dispersed mixture, 45 g of purified water was added to obtain 50 gof a suspension. The obtained suspension was measured for the particlesize distributions using a particle size distribution analyzer, and theparticle size distributions of clobetasol propionate were found to havea mean particle diameter (Dv) of 637 nm, a 10% particle diameter (D10)of 233 nm, a median particle diameter (D50) 475 nm and a 90% particlediameter (D90) of 1129 nm.

(Example 5) Preparation of an Ophthalmic Suspension ContainingClobetasol Propionate Nanoparticle

(1) Preparation of a 0.05% Ophthalmic Nanoparticle Suspension (a MeanParticle Diameter of about 100 nm)

Into a beaker, 2.4 g of the pulverized-kneaded product (dough) producedin Example 4 (1), 150 g of a 0.01% UNILUB aqueous solution and 30 g of a1.0% PVA (Merck KGaA) aqueous solution were weighed, and homogeneouslydispersed for about 5 minutes using a ultrasonic device (MODELVS-100III, AS ONE Corporation) to give a crude dispersion, which wasprocessed using a high pressure homogenizer (SANWA ENGINEERING LTD.,L01-YH1) to obtain a dispersion. To the dispersion, 2.5 g of a 0.1%benzalkonium chloride (BAC) aqueous solution and 2.5 g of a 3.0%hydroxypropyl methylcellulose (HPMC) aqueous solution were added, towhich subsequently a 500 mM sodium citrate was gradually added up to pH7.0. Water for injection was then added thereto to give a total amountof 417.6 g to obtain a 0.05% ophthalmic nanosuspension (a mean particlediameter of about 100 nm). The obtained ophthalmic suspension had anosmotic pressure ratio of 0.8.

(2) Preparation of a 0.05% Ophthalmic Nanosuspension (a Mean ParticleDiameter of about 300 nm)

Into a beaker, 2.1 g of the pulverized-kneaded product (dough) producedin Example 4 (3), 150 g of a 0.01% UNILUB aqueous solution and 30 g of a1.0% PVA aqueous solution were weighed, and homogeneously dispersed forabout 5 minutes using a ultrasonic device (MODEL VS-100III, AS ONECorporation) to give a crude dispersion, which was processed using ahigh pressure homogenizer (SANWA ENGINEERING LTD., L01-YH1) to obtain adispersion. To the dispersion, 2.5 g of a 0.1% BAC aqueous solution and2.5 g of a 3.0% HPMC aqueous solution were added, to which subsequentlya 500 mM sodium citrate was gradually added up to pH 7.0. Water forinjection was then added to give a total amount of 405.4 g to obtain a0.05% ophthalmic nanosuspension (a mean particle diameter of about 300nm). The obtained ophthalmic suspension had an osmotic pressure ratio of0.8.

(3) Preparation of a 0.05% Ophthalmic Nanosuspension (a Mean ParticleDiameter of about 600 nm)

Into a beaker, 0.52 g of the pulverized-kneaded product (dough) producedin Example 4 (4), 150 g of water for injection and 30 g of a 1.0% PVAaqueous solution were weighed, and homogeneously dispersed for about 5minutes using a ultrasonic device (MODEL VS-100III, AS ONE Corporation)to give a crude dispersion, which was processed using a high pressurehomogenizer (SANWA ENGINEERING LTD., L01-YH1) to obtain a dispersion. Tothe dispersion, 2.5 g of a 0.1% BAC aqueous solution and 2.5 g of a 3.0%HPMC aqueous solution were added, to which subsequently a 500 mM sodiumcitrate was gradually added up to pH 7.0. To the obtained mixture, 1.45g of sodium chloride was added, and then water for injection was addedto give a total amount of 245 g to obtain a 0.05% ophthalmicnanosuspension (a mean particle diameter of about 600 nm). The obtainedophthalmic suspension had an osmotic pressure ratio of 0.9.

Table 6 shows the composition of each of the 0.05% clobetasol propionateophthalmic nanosuspensions prepared in Examples 5 (1) to (3).

TABLE 6 Composition (mg/mL) Component (1) (2) (3) Clobetasol propionate0.5 0.5 0.5 Sodium chloride 5.5 5.5 5.9 Hydrogenated soybean lecithin0.5 — — Glycerin 0.86 0.86 0.5 Anhydrous citric acid 0.04 0.04 0.04 PVA1 1 1 POE•POP glycol 0.05 0.05 — BAC 0.01 0.01 0.01 HPMC 3 3 3 Sodiumcitrate quantum quantum quantum sufficit sufficit sufficit Water forinjection quantum quantum quantum sufficit sufficit. sufficit

(Example 6) Intraocular Pharmacokinetics Test

The ophthalmic nanosuspensions prepared in Examples 5(1) to 5(3) wereophthalmically administered into the eyes of rabbits (Kbl:JW, male) totest an intraocular pharmacokinetics (n=3). The lower eyelid of eachrabbit was gently pulled off, the test substance was ophthalmicallyadministered into the conjunctival sac of the left eye using a pipette(single ocular administration, 50 μL/eye), and the upper and lowereyelids were gently closed after administration and held for about 2seconds. After 15 minutes, 30 minutes, 60 minutes, and 90 minutes fromthe administration, the rabbits were anesthetized by administering anaqueous solution of pentobarbital sodium (Tokyo Chemical Industry Co.,Ltd.) via their auricular veins and then euthanized by bleeding. Eyeswere thoroughly washed with water for injection, the aqueous humor (lefteye) was collected, and subsequently the conjunctiva (left eye) wascollected. Each of the collected aqueous humor and conjunctiva wasweighed by an electronic force balance, and then frozen by liquidnitrogen, which was stored in an ultracold freezer (acceptable range:−70° C. or lower) until measurement. The clobetasol propionateconcentrations in aqueous humor and conjunctiva were measured byLC-MS/MS.

(Pretreatment of Aqueous Humor)

To 25 μL of the collected aqueous humor, 20 μL of methanol and 20 μL ofa solution of an internal standard substance (prednisolone) were addedand thoroughly stirred. To the resulting mixture, 100 μL of acetonitrilewas added and thoroughly stirred. After centrifugation (13100×g, 4° C.,5 minutes), 10 μL of the supernatant was injected into the LC-MS/MS.

(Pretreatment of Conjunctiva)

To the collected conjunctiva, ultrapure water was added in a nine timesvolume of the wet weight of the conjunctiva, and homogenized. To 25 μLof the homogenate, 25 μL of methanol and 20 μL of a solution of aninternal standard substance (prednisolone) were added, and thoroughlystirred. To the resulting mixture, 100 μL of acetonitrile was added, andthoroughly stirred. After centrifugation (13100×g, 4° C., 5 minutes), 20μL of the supernatant was injected into the LC-MS/MS.

(Measurement Conditions in LC-MS/MS)

(Measurement Conditions in HPLC)

Column: CAPCELL PAK C18 MGIII (5 μm, 2 mm×150 mm, Shiseido Company,Limited)

Mobile Phase A: 0.2% aqueous solution of formic acid

Mobile Phase B: Acetonitrile

Gradient Time Program: The following volume ratios were employed.

Time (min) Mobile Phase A (%) Mobile Phase B (%) 0.00 70 30 2.20 70 302.50 20 80 5.40 20 80 5.41 70 30 7.00 70 30Flow Rate: 0.3 mL/minColumn Temperature: 40° C.Autosampler Temperature: 4° C.Analysis Time: 7 minutes(Measurement Conditions in MS/MS)Ion Source: Electrospray ionization (ESI)Scan Type: Multiple reaction monitoring (MRM)Polarity: PositiveSource Temperature: 400° C.Monitored Ions:

Compounds Q1 (m/z) Q3 (m/z) Clobetasol Propionate 468.1 356.3 InternalStandard Substance 361.3 147.1 (Prednisolone)Acceptable Range: Within ±0.5

As the results of the intraocular pharmacokinetics test of theophthalmic nanosuspensions prepared in Examples 5(1) to 5(3), the timecourse changes of the drug concentration in aqueous humor are shown inFIG. 1 and Table 7, and the time course changes of the drugconcentration in conjunctiva are shown in FIG. 2 and Table 8. The drugconcentration in aqueous humor indicated particle diameter dependence.The drug concentration in aqueous humor tends to increase withdecreasing particle diameter. Thus, it is shown that a smaller particlediameter is more suitable for achieving better migration of theophthalmically administered nano-sized clobetasol propionate intoaqueous humor. The drug concentration in conjunctiva also showed a trendfor particle diameter dependence, which indicated that a smallerparticle diameter is more suitable for transferability of theophthalmically administered nano-sized clobetasol propionate intoconjunctiva.

TABLE 7 Concentration (ng/mL) 0.05% ophthalmic 0.05% ophthalmic 0.05%ophthalmic nanosuspension nanosuspension nanosuspension (mean particle(mean particle (mean particle Time diameter = 600 nm) diameter = 300 nm)diameter = 100 nm) (min) Mean SD Mean SD Mean SD 15 0 0 14.00 1.07 23.1920.61 30   9.27   2.67 24.45 13.76  39.92  7.65 60   6.45   1.24 25.926.51 75.32 29.8  90   5.45   1.62 20.80 8.20 23.19  1.04

TABLE 8 Concentration (ng/mL) 0.05% ophthalmic 0.05% ophthalmic 0.05%ophthalmic nanosuspension nanosuspension nanosuspension (mean particle(mean particle (mean particle Time diameter = 600 nm) diameter = 300 nm)diameter = 100 nm) (min) Mean SD Mean SD Mean SD 15 255   74.5 703.4148.7 1210   391   30 149.1  140   640.0 793.3 203.4 34.3 60  73.34 24.01  88.98  32.6 429.4 68.4 90  5.32  9.22  78.25  43.99  95.96 20.43

(Example 7) Examination of Influence of Thickener on OphthalmicNanosuspension

Since Example 6 showed that the mean particle diameter of the nano-sizedclobetasol propionate is suitably about 100 nm, using ophthalmicsuspensions containing nano-sized clobetasol propionate with a meanparticle diameter of about 100 nm, an intraocular pharmacokinetics weretested for various viscosities of ophthalmic nanosuspensions which werecontrolled by employing various thickeners.

(1) Preparation of Ophthalmic Nanosuspension P

Into a beaker, 5 g of the pulverized-kneaded product (dough) produced inExample 4(2), 335 g of a 0.01% aqueous solution of UNILUB, and 67 g of a1.0% aqueous solution of PVA were weighed, which were homogeneouslydispersed for about 5 minutes using an ultrasonic device (MODELVS-100III, AS ONE Corporation) to give a crude dispersion. The crudedispersion was processed by a high-pressure homogenizer (L01-YH1, SANWAENGINEERING LTD.) to obtain a dispersion. To the dispersion, 6.7 g of a0.1% aqueous solution of BAC and 201 g of an aqueous solution of 1.0%HPMC (60SH-50) were added, and then 500 mM sodium citrate was graduallyadded to adjust the pH to 7.0. Subsequently, water for injection wasadded to give a total amount of 670 g to obtain a 0.05% ophthalmicnanosuspension P. The viscosity of the ophthalmic suspension was about 2mPa·S.

(2) Preparation of Ophthalmic Nanosuspension Q

A 0.05% ophthalmic nanosuspension Q was prepared in the same manner asExample 7(1), except for substituting “100.5 g of an aqueous solution of1.0% HPMC (60SH-4000)” for “201 g of an aqueous solution of 1.0% HPMC(60SH-50)”. The viscosity of the ophthalmic suspension was about 3mPa·S.

(3) Preparation of Ophthalmic Nanosuspension R

A 0.05% ophthalmic nanosuspension R was prepared in the same manner asExample 7(1), except for substituting “134 g of an aqueous solution of1.0% MC (SM-100)” for “201 g of an aqueous solution of 1.0% HPMC(60SH-50)”. The viscosity of the ophthalmic suspension was about 2mPa·S.

(4) Preparation of Ophthalmic Nanosuspension S

A 0.05% ophthalmic nanosuspension S was prepared in the same manner asExample 7(1), except for substituting “100.5 g of an aqueous solution of1.0% MC (SM-4000)” for “201 g of an aqueous solution of 1.0% HPMC(60SH-50)”. The viscosity of the ophthalmic suspension was about 3mPa·S.

The compositions of the 0.05% clobetasol propionate ophthalmicnanosuspensions prepared in Examples 7(1) to 7(4) are shown in thefollowing Table 9.

TABLE 9 Composition (mg/mL) Example 7 (1) (2) (3) (4) Ophthalmic P Q R Snanosuspension Clobetasol propionate 0.5 0.5 0.5 0.5 Sodium chloride 5.55.5 5.5 5.5 Hydrogenated soybean 0.5 0.5 0.5 0.5 lecithin Glycerin 0.840.84 0.84 0.84 Anhydrous citric acid 0.04 0.04 0.04 0.04 PVA 1 1 1 1POE-POP glycol 0.05 0.05 0.05 0.05 BAC 0.01 0.01 0.01 0.01 HPMC(60SH-50) 3 — — — HPMC (60SH-4000) — 1.5 — — MC (SM-100) — — 2 — MC(SM-4000) — — — 1.5 Sodium citrate quantum quantum quantum quantumsufficit sufficit sufficit sufficit Water for injection quantum quantumquantum quantum sufficit sufficit sufficit sufficit(5) Intraocular Pharmacokinetics Test

The ophthalmic nanosuspensions prepared in Examples 7(1) to 7(4) weresubjected to an intraocular pharmacokinetics test according to themethod described in Example 6.

(6) Results

The time course changes of the drug concentration in aqueous humor areshown in FIG. 3 and Table 10, and the time course changes of the drugconcentration in conjunctiva are shown in FIG. 4 and Table 11. Theresults shown in FIG. 3 demonstrated that higher viscosity of anophthalmic suspension showed better transferability of the ophthalmicsuspension into aqueous humor. The results shown in FIG. 4 demonstratedthat higher viscosity of an ophthalmic suspension showed bettertransferability of the ophthalmic suspension into conjunctiva in theinitial phase (in the first 15 minutes).

TABLE 10 Concentration (ng/mL) 0.05% 0.05% 0.05% 0.05% ophthalmicophthalmic ophthalmic ophthalmic nano- nano- nano- nano- Time suspensionP suspension Q suspension R suspension S (min) Mean SD Mean SD Mean SDMean SD 15 25.77 8.94 24.83 11.92  17.03 2.16 23.49 1.43 30 63.88 12.87 74.62 5.81 54.74 12.37  63.16 2.18 60 48.58 27.62  53.09 15.86  52.6910.34  52.08 17.4  90 36.67 11.72  54.23 38.82  33.8  13.71  59.3428.99 

TABLE 11 Concentration (ng/mL) 0.05% ophthalmic 0.05% ophthalmic 0.05%ophthalmic 0.05% ophthalmic Time nanosuspension P nanosuspension Qnanosuspension R nanosuspension S (min) Mean SD Mean SD Mean SD Mean SD15 971.9  162.8  1455.00 641.00 659.10 174.50 2141  708  30 608.40228.10  525.70  5.10 367.7   37.20   497.3   59.6 60 260.90 182.30 204.80  42.90 269.9  101.90   177.2   86.4 90 127.00  36.80  288.20198.10 199.9   88.10   148.4 48

(Example 8) Efficacy of Clobetasol Ophthalmic Nanosuspension for RabbitModel of BSA-Induced Uveitis

(1) Pulverization of Clobetasol Propionate

Clobetasol propionate was pulverized in the same manner as Example 4(1)to produce a pulverized-kneaded product (dough) containing clobetasolpropionate particles with a particle size distribution having the meanparticle diameter (Dv) of 132 nm, the 10% particle diameter (D10) of 65nm, the median particle diameter (D50) of 109 nm, and the 90% particlediameter (D90) of 186 nm.

(2) Preparation of 0.05% Clobetasol Propionate Ophthalmic Nanosuspension

Into a beaker, 2.4 g of the pulverized kneaded product (dough) preparedin (1) above, 167.5 g of a 0.01% aqueous solution of POE⋅POP glycol, and33.5 g of a 1.0% aqueous solution of PVA were weighed, and dispersedusing an ultrasonic device (MODEL VS-100III, AS ONE Corporation) to givea crude dispersion. The crude dispersion was processed by ahigh-pressure homogenizer (L01-YH1, SANWA ENGINEERING LTD.) five timesto obtain a dough dispersion. To the dispersion, 2.8 g of a 0.1% aqueoussolution of benzalkonium chloride and 56.4 g of a 1.0% aqueous solutionof methyl cellulose were added, and then a 500 mM aqueous solution ofsodium citrate was gradually added to adjust the pH to 7.0. Then, 1.5 gof glycerin was added to adjust the osmotic pressure ratio to 1.0, andwater for injection was added to give 282.1 g in a total amount of a0.05% clobetasol propionate ophthalmic nanosuspension. The compositionand physical properties of the ophthalmic suspension are shown in thefollowing tables.

Composition of Ophthalmic Suspension

Components Composition (%) Clobetasol Propionate 0.05 Sodium Chloride0.50 Hydrogenated Soybean Lecithin 0.05 Glycerin 0.08 Anhydrous CitricAcid 0.004 Polyoxyethylene Polyoxypropylene Glycol 0.005 PolyvinylAlcohol 0.1 Benzalkonium Chloride 0.001 Methyl Cellulose 0.20 SodiumCitrate quantum sufficit Water for Injection quantum sufficitPhysical Properties of Ophthalmic Suspension

Measurement Items Measured Values Clobetasol Propionate Concentration(%) 0.05 Osmotic Pressure Ratio 1.0 pH 7.0 Viscosity (mPa · S) 2.1(3) Efficacy Using Rabbit Model of BSA-Induced Uveitis

Rabbits (Std:JW/CSK) were anesthetized by a combination of ketaminehydrochloride (500 mg of Ketalar for intramuscular injection) andxylazine (2% Celactal injection), and 0.4% oxybuprocaine hydrochloride(Benoxil ophthalmic solution 0.4%) was ophthalmically administered intothe right eyeball of each rabbit to anesthetize. After loss of cornealreflex, 0.1 mL of a 10% physiological saline solution of BSA wasinjected into the central region of the vitreous body of the right eyeto cause uveitis (the first induction). From the next day, 50 μL of acontrol substance (physiological saline solution), 50 μL of the testsubstance (the 0.05% clobetasol propionate ophthalmic nanosuspensionprepared in (2) above), and 50 μL of a positive control substance(commercially-available 0.1% fluorometholone ophthalmic solution) wereeach weighed with a micropipette and administered into the righteyeballs twice a day (at 9:00 and 17:00 in principle) for 29 consecutivedays. The left eyes were untreated, and n=5 for each group.

During 4 days from the 15th to 18th day after the first administrationof BSA, the symptoms of inflammation of the external eye (the outside ofthe cornea) and the internal eye (the inside of the cornea) were scoredaccording to the ocular inflammation grading criteria specified byYamauchi et al. (Hideyasu Yamauchi et al. (1973), Folia ophthalmologicaJaponica, 24, 969-979) to evaluate the anti-inflammatory effect. On the27th day, a physiological saline solution of 1.25% BSA was injected viathe auricular veins at a dose of 2 mL/kg to cause uveitis (the secondinduction). On the 29th day, the symptoms of inflammation of theexternal and internal eyes were scored in the same manner as describedabove to evaluate the anti-inflammatory effect.

(4) Results

The results are shown in FIGS. 5 to 7, which demonstrated that the 0.05%clobetasol propionate ophthalmic nanosuspension has the sameanti-inflammatory effect on the inflammation model of external andinternal eyes as the 0.1% fluorometholone ophthalmic solution.

(Example 9) Efficacy for Rat Model of Croton-Induced Conjunctivitis

(1) Preparation of 0.1% Clobetasol Propionate Ophthalmic Nanosuspension

Into a beaker, 4.2 g of the pulverized-kneaded product (dough) preparedin Example 8(1) above, 150 g of a 0.01% aqueous solution ofpolyoxyethylene polyoxypropylene glycol, and 30 g of a 1.0% aqueoussolution of PVA were weighed, and were dispersed using an ultrasonicdevice (MODEL VS-100III, AS ONE Corporation) to give a crude dispersion.The crude dispersion was processed by a high-pressure homogenizer(L01-YH1, SANWA ENGINEERING LTD.) five times to obtain a doughdispersion. To the dispersion, 2.4 g of a 0.1% aqueous solution ofbenzalkonium chloride and 48.3 g of a 1.0% aqueous solution of methylcellulose were added, and then a 500 mM aqueous solution of sodiumcitrate was gradually added to adjust the pH to 7.0. Then, water forinjection was added to give 241.4 g in total amount of 0.1% clobetasolpropionate ophthalmic nanosuspension. The composition and physicalproperties of the ophthalmic suspension are shown in the followingtables.

Composition of Ophthalmic Suspension

Components Composition (%) Clobetasol Propionate 0.1 Sodium Chloride 1.1Hydrogenated Soybean Lecithin 0.1 Glycerin 0.16 Anhydrous Citric Acid0.008 Polyoxyethylene Polyoxypropylene Glycol 0.005 Polyvinyl Alcohol0.1 Benzalkonium Chloride 0.001 Methyl Cellulose 0.20 Sodium Citratequantum sufficit Water for Injection quantum sufficitPhysical Properties of Ophthalmic Suspension

Measurement Items Measured Values Clobetasol Propionate Concentration(%) 0.1 Osmotic Pressure Ratio 1.6 pH 7.0 Viscosity (mPa · S) 1.9(2) Efficacy Using Rat Model of Croton-Induced Conjunctivitis

Ethanol (inflammatory agent) was ophthalmically administered into botheyes of rats (Wistar, female) at a dose of 2.5 μL/site to causeinflammation at −41 minutes and at 0 minute, twice in total. The testsubstance (0.1% clobetasol propionate ophthalmic nanosuspension preparedin (1)) and a positive control substance (commercially-available 0.1%dexamethasone) were ophthalmically administered into both eyes of therats with a micropipette at a dose of 5 μL/site twice, 1 minute beforethe first administration of the inflammatory agent (at −42 minutes) and1 minute before the second administration of the inflammatory agent (at−1 minute). A normal control group (not caused inflammation without drugadministration) and an inflammation control group (caused inflammationwithout drug administration) were used as control groups, and n=10 foreach group.

A 10% ethanol solution of croton oil (inflammation-inducing agent) wasophthalmically administered into both eyes of the rats at a dose of 5μL/site to induce inflammation three times in total, 40 minutes after,100 minutes after, and 160 minutes after the second administration ofthe inflammatory agent. After 2 hours from the last administration ofthe 10% ethanol solution of croton oil, the rats were euthanized bycervical dislocation under isoflurane anesthesia, and then theconjunctiva was collected from both eyes. The weight of the conjunctivawas measured. The anti-inflammatory effect of the test substance wasevaluated from the conjunctival weight comparing with the conjunctivalweight of the inflammation control group.

The results are shown in FIG. 8, which show that the conjunctival weightof the inflammation control group was greater than that of the normalcontrol group, and thus inflammation was confirmed to be induced in themodel. The conjunctival weights of both of the groups in which the testsubstance (0.1% clobetasol propionate ophthalmic nanosuspension) wasadministered and in which the positive control substance (0.1%dexamethasone) was administered were smaller than that of theinflammation control group. Thus, the 0.1% clobetasol propionateophthalmic nanosuspension of the present application was shown to beable to suppress edema of the conjunctiva in ophthalmicallyadministering into the eyes of rats model of croton-inducedconjunctivitis.

(Example 10) Efficacy for Rat Model of Carrageenan-Induced ConjunctivalEdema

(1) Preparation of 0.1% Clobetasol Propionate Ophthalmic Nanosuspension

Into a beaker, 4.3 g of the pulverized kneaded product (dough) preparedin Example 8(1) above, 150 g of a 0.01% aqueous solution ofpolyoxyethylene polyoxypropylene glycol, and 30 g of a 1.0% aqueoussolution of PVA were weighed, and were dispersed using an ultrasonicdevice (MODEL VS-100III, AS ONE Corporation) to give a crude dispersion.The crude dispersion was processed by a high-pressure homogenizer(L01-YH1, SANWA ENGINEERING LTD.) five times to obtain a doughdispersion. To the dispersion, 2.4 g of a 0.1% aqueous solution ofbenzalkonium chloride and 47.9 g of a 1.0% aqueous solution of methylcellulose were added, and then a 500 mM aqueous solution of sodiumcitrate was gradually added to adjust the pH to 7.0. Then, water forinjection was added to give 239.5 g in a total amount of 0.1% clobetasolpropionate ophthalmic nanosuspension. The composition and physicalproperties of the ophthalmic suspension are shown in the followingtables.

Composition of Ophthalmic Suspension

Components Composition (%) Clobetasol Propionate 0.1 Sodium Chloride 1.0Hydrogenated Soybean Lecithin 0.1 Glycerin 0.16 Anhydrous Citric Acid0.008 Polyoxyethylene Polyoxypropylene Glycol 0.005 Polyvinyl Alcohol0.1 Benzalkonium Chloride 0.001 Methyl Cellulose 0.20 Sodium Citratequantum sufficit Water for Injection quantum sufficitPhysical Properties of Ophthalmic Suspension

Measurement Items Measured Values Clobetasol Propionate Concentration(%) 0.1 Osmotic Pressure Ratio 1.5 pH 7.0 Viscosity (mPa · S) 1.9(2) Efficacy Using Rat Model of Carrageenan-Induced Conjunctival Edema

A control substance (physiological saline solution), the test substances(the 0.05% clobetasol propionate ophthalmic nanosuspension prepared inExample 8(2) and the 0.1% clobetasol propionate ophthalmicnanosuspension prepared in Example 10(1)), and a positive controlsubstance (commercially-available 0.1% fluorometholone ophthalmicsolution) were administered into the right eyes of rats (Wistar, male)using a micropipette (n=8 for each group). After 15 minutes from theophthalmic administration, 50 μL of a physiological saline solution of1% carrageenan (inflammatory substance) was subcutaneously administeredinto the right upper palpebral conjunctiva of the rats under isofluraneanesthesia to generate a conjunctival edema model. After 4 hours fromthe administration of the inflammatory substance, the rats wereeuthanized by bleeding from abdominal aorta under isoflurane anesthesia,and each edematous area including the right eyeball and accessorylacrimal glands (Harderian glands) was isolated. The right palpebralconjunctiva was then separated from the edematous area, and the weightof the conjunctiva was measured. The determined palpebral conjunctivalweights were compared to evaluate the anti-inflammatory effect.

The results of the measurement of the palpebral conjunctival weight areshown in FIG. 9, which demonstrated the concentration dependentanti-inflammatory effect of the clobetasol propionate ophthalmicnanosuspension, and showed that the 0.1% clobetasol propionateophthalmic nanosuspension exhibits substantially the same degree ofanti-inflammatory activity as the positive control substance, the 0.1%fluorometholone ophthalmic solution.

(Example 11) Efficacy of Clobetasol Ophthalmic Nanosuspension for RabbitModel of LPS-Induced Uveitis

(1) Pulverization of Clobetasol Propionate

In a 1.0 L water-cooled vertical kneader (manufactured by INOUE MFG.,INC.), 50 g of clobetasol propionate (FARMABIOS S.p.A.), 550 g of sodiumchloride (Tomita Salt K-30, Tomita Pharmaceutical Co., Ltd.), 4 g ofanhydrous citric acid (Sigma-Aldrich Co. LLC.), and 50 g of hydrogenatedsoybean lecithin (Phospholipon 90H, Lipoid GmbH) were added, and werehomogeneously mixed. To the mixture, 70 g of glycerin (Sigma-Aldrich Co.LLC.) was added with keeping the mixture in form of dough, andpulverized at 5° C. for 5 hours. The resulting pulverized-kneadedproduct (dough) was dispersed using a dispersant to give a suspension inthe same manner as Example 1(1), and the particle size distribution ofthe clobetasol propionate was measured. The particle size distributionsof clobetasol propionate were found to have the mean particle diameter(Dv) of 132 nm, the 10% particle diameter (D10) of 67 nm, the medianparticle diameter (D50) of 110 nm, and the 90% particle diameter (D90)of 184 nm.

(2) Preparation of 0.002% Clobetasol Propionate OphthalmicNanosuspension

Into a beaker, 0.076 g of the pulverized-kneaded product (dough)prepared in (1), 31.3 g of a 0.01% aqueous solution of Poloxamer 407,25.0 g of a 1.0% aqueous solution of PVA, 0.217 g of sodium chloride,and 93.3 g of water for injection were weighed, and were dispersed usingan ultrasonic device to give a crude dispersion. The crude dispersionwas processed by a high-pressure homogenizer (L01-YH1, SANWA ENGINEERINGLTD.) four times to obtain a dough dispersion. Into a beaker, 110.67 gof the dough dispersion was weighed, to which 1.85 g of a 0.1% aqueoussolution of benzalkonium chloride and 36.91 g of a 1.0% aqueous solutionof methyl cellulose were added. A 1 M aqueous solution of sodium citratewas then gradually added to adjust the pH to 7.0. Then, glycerin wasadded to adjust the osmotic pressure ratio to 1.0, and water forinjection was added to give 184.6 g in a total amount of 0.002%clobetasol propionate ophthalmic nanosuspension. The composition andphysical properties of the ophthalmic suspension are shown in thefollowing tables.

Composition of Ophthalmic Suspension

Components Composition (%) Clobetasol Propionate 0.002 Sodium Chloride0.11 Hydrogenated Soybean Lecithin 0.002 Glycerin 2.2 Anhydrous CitricAcid 0.0002 Poloxamer 407 0.0013 Polyvinyl Alcohol 0.1 BenzalkoniumChloride 0.001 Methyl Cellulose 0.20 Sodium Citrate quantum sufficitWater for Injection quantum sufficitPhysical Properties of Ophthalmic Suspension

Measurement Items Measured Values Clobetasol Propionate Concentration(%) 0.002 Osmotic Pressure Ratio 1.0 pH 7.0 Viscosity (mPa · S) 1.98(3) Preparation of 0.01% Clobetasol Propionate Ophthalmic Nanosuspension

Into a beaker, 0.38 g of the pulverized-kneaded product (dough) preparedin (1), 62.5 g of a 0.01% aqueous solution of Poloxamer 407, 25.0 g of a1.0% aqueous solution of PVA, and 62.5 g of water for injection wereweighed, and were dispersed using an ultrasonic device to give a crudedispersion. The crude dispersion was processed by a high-pressurehomogenizer (L01-YH1, SANWA ENGINEERING LTD.) four times to obtain adough dispersion. Into a beaker, 119.44 g of the dough dispersion wasweighed, to which 1.98 g of a 0.1% aqueous solution of benzalkoniumchloride and 39.70 g of a 1.0% aqueous solution of methyl cellulose wereadded. A 1 M aqueous solution of sodium citrate was then gradually addedto adjust the pH to 7.0. After that, glycerin was added to adjust theosmotic pressure ratio to 1.0, and water for injection was added to give198.5 g in total amount of 0.01% clobetasol propionate ophthalmicnanosuspension. The composition and physical properties of theophthalmic suspension are shown in the following tables.

Composition of Ophthalmic Suspension

Components Composition (%) Clobetasol Propionate 0.01 Sodium Chloride0.12 Hydrogenated Soybean Lecithin 0.01 Glycerin 2.1 Anhydrous CitricAcid 0.0008 Poloxamer 407 0.0025 Polyvinyl Alcohol 0.1 BenzalkoniumChloride 0.001 Methyl Cellulose 0.20 Sodium Citrate quantum sufficitWater for Injection quantum sufficitPhysical Properties of Ophthalmic Suspension

Measurement Items Measured Values Clobetasol Propionate Concentration(%) 0.010 Osmotic Pressure Ratio 1.0 pH 7.0 Viscosity (mPa · S) 1.99(4) Preparation of 0.05% Clobetasol Propionate Ophthalmic Nanosuspension

Into a beaker, 1.84 g of the pulverized kneaded product (dough) preparedin (1), 125.0 g of a 0.01% aqueous solution of Poloxamer 407, and 25.0 gof a 1.0% aqueous solution of PVA were weighed, and were dispersed usingan ultrasonic device to give a crude dispersion. The crude dispersionwas processed by a high-pressure homogenizer (L01-YH1, SANWA ENGINEERINGLTD.) four times to obtain a dough dispersion. Into a beaker, 116.79 gof the dough dispersion was weighed, to which 1.92 g of a 0.1% aqueoussolution of benzalkonium chloride and 38.45 g of a 1.0% aqueous solutionof methyl cellulose were added. A 1 M aqueous solution of sodium citratewas then gradually added to adjust the pH to 7.0. Then, glycerin wasadded to adjust the osmotic pressure ratio to 1.0, and water forinjection was added to give 192.3 g in total amount of 0.05% clobetasolpropionate ophthalmic nanosuspension. The composition and physicalproperties of the ophthalmic suspension are shown in the followingtables.

Composition of Ophthalmic Suspension

Components Composition Ratio (%) Clobetasol Propionate 0.05 SodiumChloride 0.56 Hydrogenated Soybean Lecithin 0.05 Glycerin 0.50 AnhydrousCitric Acid 0.004 Poloxamer 407 0.005 Polyvinyl Alcohol 0.1 BenzalkoniumChloride 0.001 Methyl Cellulose 0.20 Sodium Citrate quantum sufficitWater for Injection quantum sufficitPhysical Properties of Ophthalmic Suspension

Measurement Items Measured Values Clobetasol Propionate Concentration(%) 0.048 Osmotic Pressure Ratio 1.0 pH 7.0 Viscosity (mPa · S) 1.99(5) Efficacy for Rabbit Model of LPS-Induced Uveitis

Rabbits (Kbs:JW) were anesthetized by administering pentobarbital sodium(Somnopentyl) via their auricular veins, and 0.4% oxybuprocainehydrochloride (Benoxil ophthalmic solution) was then ophthalmicallyadministered into both eyes of the rabbits. After loss of cornealreflex, a lid speculum was attached to each rabbit, and 0.02 mL of LPS(Lipopolysaccharide, from E. coli O55: sigma) adjusted to aconcentration of 2 μg/mL was administered into the vitreous body using asyringe with a 30G needle to cause inflammation. A control substance(saline), a positive control substance (durezol (registered trademark):0.05% difluprednate ophthalmic emulsion manufactured by AlconLaboratories Inc.), and the test substance (0.05% ophthalmic suspension)prepared in (4) above were ophthalmically administered into both eyes ofthe rabbits using a micropipette at a dose of 50 μL, 4 hours before, 15minutes after, 6 hours after, and 8 hours after the LPS administration.For each group, both eyes of 6 rabbits were used so as to set n=12 foreach group. At 24 hours after the LPS administration, the rabbits wereeuthanized by excessive administration of pentobarbital sodium(Somnopentyl), and the whole amount of anterior chamber aqueous humorwas collected using a syringe with a 26G needle. The eyeballs wereisolated and incised around the corneoscleral limbus, and the vitreousbody was collected using a 1 mL syringe. The PGE2 concentrations in thecollected samples of both anterior chamber aqueous humor and vitreousbody were measured by ELISA assay (Prostaglandin E2 Express ELISA Kit:cayman).

(6) Results

FIG. 10 shows the results of the measurement of the PGE2 concentrationin aqueous humor (evaluation of anterior eye segment), and FIG. 11 showsthe results of the measurement of the PGE2 concentration in vitreousbody (evaluation of posterior eye segment). These results demonstratedthat when ophthalmically administered into the eyes of rabbits model ofLPS-induced uveitis, the clobetasol propionate ophthalmic nanosuspensionof the present invention exhibits the same level of anti-inflammatoryaction on uveitis (anterior eye segment) as the positive control,Durezol (registered trademark). Additionally, the PGE2 concentration invitreous body was lower in the group administered the clobetasolpropionate ophthalmic nanosuspension of the present invention than forthe group administered Durezol (registered trademark), whichdemonstrated that the clobetasol propionate ophthalmic nanosuspension ofthe present invention exhibits higher anti-inflammatory activity onuveitis (posterior eye segment) than Durezol (registered trademark).

(Example 12) Efficacy of Clobetasol Ophthalmic Nanosuspension for RabbitModel of Puncture-Induced Anterior Chamber Inflammation

(1) Efficacy for Rabbit Model of Puncture-Induced Anterior ChamberInflammation

A control substance (saline), a positive control substance (Durezol(registered trademark)), and the test substances prepared in Examples11(2), 11(3), and 11(4) (0.002%, 0.01%, and 0.05% ophthalmicsuspensions) were administered into both eyes of rabbits (Kbs:JW) usinga micropipette once at a dose of 50 μL. For each group, both eyes of 6rabbits were used so as to set n=12 for each group. After 4 hours fromthe administration, 0.4% oxybuprocaine hydrochloride (Benoxil ophthalmicsolution) was ophthalmically administered into both eyes of the rabbits.After loss of corneal reflex, a lid speculum was attached to eachrabbit, and a syringe with a 26G needle was inserted into the anteriorchamber to collect the whole amount of anterior chamber aqueous humorand thus cause inflammation of the anterior eye segment. After 3 hours,the whole amount of anterior chamber aqueous humor was collected againusing a syringe with a 26G needle, and the protein concentration inanterior chamber aqueous humor was measured by BCA assay (Pierce™ BCAProtein Assay Kit: Thermo Fisher Scientific Inc.). The proteinconcentration in anterior chamber aqueous humor was measured by BCAassay also for the group (Normal) in which no anterior eye inflammationwas caused by anterior chamber puncture.

(2) Results

FIG. 12 shows the results of the measurement of the proteinconcentration in anterior chamber aqueous humor, which demonstrated thatthe clobetasol propionate ophthalmic nanosuspensions (0.002%, 0.01%, and0.05%) of the present invention exhibit the same level ofanti-inflammatory action as the positive control, Durezol (registeredtrademark) (0.05% difluprednate) in ophthalmically administering intothe eyes of rabbits model of puncture-induced anterior chamberinflammation.

(Example 13) Efficacy of Clobetasol Ophthalmic Nanosuspension for RabbitModel of LPS-Induced Uveitis

(1) Efficacy for Rabbit Model of LPS-Induced Uveitis

Rabbits (Kbs:JW) were anesthetized by administering pentobarbital sodium(Somnopentyl) via their auricular veins, and 0.4% oxybuprocainehydrochloride (Benoxil ophthalmic solution) was then ophthalmicallyadministered into both eyes of the rabbits. After loss of cornealreflex, a lid speculum was attached to each rabbit, into the vitreousbody of which 0.02 mL of LPS (Lipopolysaccharide, from E. coli 055:sigma) adjusted to a concentration of 2 μg/mL was administered using asyringe with a 30G needle to cause inflammation. For 6 consecutive daysfrom the next day of the LPS administration, a control substance(saline), a positive control substance (Durezol: 0.05% difluprednateophthalmic emulsion manufactured by Alcon Laboratories Inc.), and thetest substance (0.05% ophthalmic suspension) prepared in Example 11(4)were ophthalmically administered into both eyes of the rabbits using amicropipette at a dose of 50 μL on a b.i.d schedule (twice dailyadministration) in which the administration was done at 9:00 and 17:00and on a q.i.d schedule (four-times daily administration) in which theadministration was done at 9:00, 12:00, 15:00, and 18:00. For eachgroup, both eyes of 4 or 5 rabbits were used so as to set n=8 or 10 foreach group. After 24 hours from the LPS administration, the rabbits wereeuthanized by excessive administration of pentobarbital sodium(Somnopentyl). Their eyeballs were isolated and incised around thecorneoscleral limbus, and the vitreous body was collected using a 1 mLsyringe. The PGE2 concentrations in the collected samples were measuredby ELISA assay (Prostaglandin E2 Express ELISA Kit: cayman).

(2) Results

FIG. 13 shows the results of the measurement of the PGE2 concentrationin vitreous body (evaluation of posterior eye segment). For the case ofthe control substance, the PGE2 concentration in vitreous body was 345.6pg/ml. The twice daily administration and four-times dailyadministration of the positive control Durezol yielded PGE2concentrations in vitreous body of 256.35 pg/ml and 179.4 pg/ml,respectively, which means that Durezol has a trend toward improvement.The twice daily administration and four-times daily administration ofthe clobetasol propionate ophthalmic nanosuspension of the presentinvention yielded PGE2 concentrations in vitreous body of 219.2 pg/mland 167.6 pg/ml, respectively, which means that the ophthalmicsuspension exhibited higher anti-inflammatory activity than Durezol. Ithas been demonstrated that the clobetasol propionate ophthalmicnanosuspension of the present invention exhibits higheranti-inflammatory action than the positive control Durezol inophthalmically administering into the eyes of rabbits model ofLPS-induced uveitis on a b.i.d schedule (twice daily administration) aswell as on a q.i.d schedule (four-times daily administration).

What is claimed is:
 1. A method of treating an eye inflammatory orinfectious disease selected from the group consisting of blepharitis,blepharoconjunctivitis, meibomitis, acute or chronic stye, chalazion,dacryocystitis, dacryoadenitis, acne rosacea of eye lid, conjunctivitis,ophthalmia neonatorum, trachoma, corneal ulcer, superficial keratitis,interstitial keratitis, keratoconjunctivitis, foreign objects,post-surgery infections of cornea, endophthalmitis, infectious uveitis,and post-surgery infections of anterior chamber and uvea, and/orreducing likelihood of ophthalmia neonatorum or infections due toblepharoplasty, chalazion removal, blepharorrhaphy, surgeries forcanaliculi and lacrimal drainage system, surgical treatments relating toeyelids and lacrimal apparatus, removal of pterygium, pinguecula ortumors, conjunctival transplant, external wounds, conjunctival flapsurgery, removal of foreign objects, keratotomy and corneal transplant,photorefractive procedure, bleb filtration, anterior chamberparacentesis, iridotomy, cataract surgery, retinal surgery, orextraocular muscle relating surgeries, said method comprisingadministering a pharmaceutical composition comprising an aqueoussuspension to a subject in need thereof in an effective amount, whereinthe aqueous suspension comprises: nanoparticles of a glucocorticosteroidcompound, wherein a mean particle diameter of the nanoparticles is 300nm or less and a D90 particle diameter of the nanoparticles is 450 nm orless, and wherein the glucocorticosteroid compound is one or moresubstances selected from the group consisting of clobetasol propionate,diflorasone diacetate, dexamethasone propionate, difluprednate,mometasone furoate, diflucortolone valerate, betamethasone butyratepropionate, fluocinonide, hydrocortisone butyrate propionate,beclomethasone dipropionate, deprodone propionate, betamethasonevalerate, dexamethasone valerate, prednisolone valerate acetate,fluocinolone acetonide, hydrocortisone butyrate, clobetasone butyrate,alclometasone dipropionate, triamcinolone acetonide, flumethasonepivalate, prednisolone and hydrocortisone; a physiologically acceptablesalt; glycerin; hydrogenated soybean lecithin; and anhydrous citricacid.
 2. The method of claim 1, wherein the nanoparticles are producedby mixing the glucocorticosteroid compound, the physiologicallyacceptable salt, glycerin, hydrogenated soybean lecithin and anhydrouscitric acid.
 3. The method of claim 2, wherein the nanoparticles areproduced by further mixing with a surface modifier.
 4. The method ofclaim 1, wherein the aqueous suspension further comprises a dispersionstabilizer.
 5. The method of claim 4, the dispersion stabilizer ispolyoxyethylene polyoxypropylene glycol and/or polyvinyl alcohol.
 6. Themethod of claim 1, wherein the aqueous suspension further comprises aviscosity modifier.
 7. The method of claim 6, the viscosity modifier isone or more substances selected from the group consisting of methylcellulose, hydroxypropyl methylcellulose and polyvinyl alcohol.
 8. Themethod of claim 6, wherein the aqueous suspension comprises 1 to 10mg/mL of the viscosity modifier.
 9. The method of claim 1, wherein thepharmaceutical composition is administered as an eye drop.